Compositions and methods for inhibiting pathogen infection

ABSTRACT

The presently-disclosed subject matter relates to antibodies, compositions, and methods for inhibiting and treating virus infection in the respiratory tract and virus transmission through the respiratory tract. In particular, the presently-disclosed subject matter relates to inhibiting and treating virus infection in a subject using compositions and antibodies that trap viruses in mucus of the respiratory tract, thereby inhibiting transport of virus across or through mucus secretions.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. provisional patentapplication No. 62/646,220, filed Mar. 21, 2018, titled “Compositionsand Methods for Inhibiting Pathogen Infection in the Lung,” which isherein incorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos.UL1TR001111 and R43AI138728, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

In particular U.S. patent application Ser. No. 14/438,511, filed Apr.24, 2015, claiming priority under 35 U.S. § 371 to PCT/US2013/067328,filed Oct. 29, 2013, titled “Compositions and Methods for InhibitingPathogen Infection”, claiming priority to U.S. provisional patentapplication No. 61/719,689, filed on Oct. 29, 2012 is hereinincorporated by reference in its entirety.

FIELD

The presently-disclosed subject matter relates to antibodies,compositions, and methods for inhibiting and treating pathogen infectionand providing contraception. In particular, the presently-disclosedsubject matter relates to inhibiting and treating pathogen infection andproviding contraception in a subject using compositions and antibodiesthat trap pathogens in mucus, thereby inhibiting transport of pathogensor sperm across or through mucus secretions. The subject matter furtherrelates to methods for monitoring the effectiveness of vaccines bydetecting antibodies capable of trapping pathogens in mucus. Forexample, the presently-disclosed subject matter relates to antibodies,compositions, and methods for inhibiting and treating virus infection inthe respiratory tract and virus transmission through the respiratorytract, including using compositions and antibodies that trap viruses inmucus of the respiratory tract, thereby inhibiting transport of virusacross or through mucus secretions.

BACKGROUND

Large quantities of IgG are transported into female genital tract mucussecretions by the MHC class I-related neonatal Fc receptor (Li et al.,Proc. Natl. Acad. Sci. U.S.A. 108:4388 (2011), resulting in at leastten-fold more IgG than IgA (Usala et al., J. Reprod. Med. 34:292(1989)). However, despite this predominance of IgG, the precisemechanism(s) by which secreted IgG can prevent vaginal infections arenot well understood. Previous animal studies have shown that antibodies(Ab) can provide robust protection against vaginal challenge withpathogens, including human immunodeficiency virus (HIV) and herpessimplex virus-2 (HSV-2), when applied intravaginally (Burton et al.,Proc. Natl. Acad. Sci. U.S.A. 108:11181 (2011); Sherwood et al., NatureBiotechnol. 14:468 (1996); Veazey et al., Nature Med. 9:343 (2003);Whaley et al., J. Infect. Dis, 169:647 (1994)) or even intravenously(Hessell et al., PLoS Pathogens 5:e1000433 (2009); Mascola et al.,Nature Med. 6:207 (2000)). Many investigators have focused onneutralizing Ab, which, at sufficiently high doses, provided sterilizingimmunity against simian-human immunodeficiency virus (SHIV) challenge inrhesus macaques (Burton et al., Proc. Natl. Acad. Sci. U.S.A. 108:11181(2011)). In the same studies, non-neutralizing or poorly neutralizing Abprovided at best only partial protection, with some infected animalsexhibiting reduced viral load. Studies by Hessell et al. and Mascola etal. showed complete protection in some animals even by weaklyneutralizing antibodies, as well as reduced viremia in others (Hessellet al., PLoS Pathogens 5:e1000433 (2009); Mascola et al., Nature Med.6:207 (2000)).

Few studies have explored the potential protective role of IgG withinthe mucus secretions overlaying the epithelial tissue, which sexuallytransmitted viruses invariably encounter and must penetrate in order toreach target cells. Well-known Ab effector functions in blood and lymph(e.g., complement activation, opsonization, and ADCC) are absent orlimited in healthy female genital secretions, which typically havelittle complement activity and few if any active leukocytes (Cone, InHandbook of Mucosal Immunology. P. L. Ogra et al., editors. AcademicPress, San Diego, Calif. 43-64 (1999); Hill et al., Am. J. Obstet.Gynecol. 166:720 (1992); Schumacher, Hum. Reprod. 3:289 (1988)). Theseclassical mechanisms of systemic immune protection do not adequatelyaccount for the moderate but significant protection observed in thelandmark Thai RV144 HIV vaccine trial (Kresge, IAVI Report Vol 13,Number 5 (2009); Rerks-Ngarm et al., N. Engl. J. Med. 361:2209 (2009)).The vaccination regimen modestly reduced the risk of HIV acquisitiondespite inducing primarily non-neutralizing Ab and otherwise offeringlittle to no protection against systemic progression of infections onceacquired, suggesting that protection likely occurred prior to initiationof infection. A better understanding of potential additional mechanismsof vaginal mucosal immunity will also likely be critical for developingeffective vaccines against other sexually transmitted infections,including HSV, which has been shown to evade complement and otherclassical antibody-mediated protective mechanisms (Brockman et al.,Vaccine 26 Suppl 8:194 (2008); Hook et al., J. Virol. 80:4038 (2006);Lubinski et al., J. Exp. Med. 190:1637 (1999)). To date, herpes vaccinecandidates have achieved only transient and partial protection despiteinducing high neutralizing serum antibody titers and cellular immunity(Chentoufi et al., Clin. Dev. Immunol. 2012:187585 (2012)). A Phase IIIclinical trial, using a subunit vaccine based on HSV-2 glycoprotein D,demonstrated some protection against HSV-1 infection but no efficacyagainst HSV-2 infection and no overall efficacy against genital disease(Belshe et al., New Eng. J. Med. 366:34 (2012)).

There is a need in the art for new compositions, and methods of usingsuch compositions, to prevent and treat infectious diseases and providecontraception.

For example, respiratory infections, such as influenza virus andrespiratory syncytial virus (RSV) infections, are a tremendous healthand financial burden. Additionally, the mucosal lining of therespiratory tract is a point of entry for non-respiratory viruses suchas Ebola virus. Ideally, viral infection in or through the lungs wouldbe prevented by transporting virions that enter the respiratory tractout of the tract before they have a chance to contact cells.

Thus, there is a need for new compositions, and methods of using suchcompositions, to prevent and treat viral infectious diseases, includingin particular diseases in the respiratory tract.

SUMMARY OF THE DISCLOSURE

Viruses must penetrate mucus to reach and infect their target cells;indeed, HIV and human papillomavirus (HPV) are both capable of rapidlydiffusing through human genital mucus secretions (Lai et al., J. Virol.83:11196 (2009); Olmsted et al., Biophys. J. 81:1930 (2001)). It waspreviously found that the diffusion of IgG (11 nm) was slowed slightlyin human cervical mucus compared to saline buffer, while much largervirus-like particles, including the capsids of norovirus (38 nm) and HPV(55 nm), were not slowed by this mucus (Olmsted et al., Biophys. J.81:1930 (2001)). It was hypothesized that the slight retardation of themuch smaller IgG molecules may be due to very transient (<1 s),low-affinity bonds with the mucin mesh (Olmsted et al., Biophys. J.81:1930 (2001)). In other words, by making only transient low-affinitybonds with mucins, IgG is able to diffuse rapidly through mucus, and intheory is therefore free to quickly accumulate on a pathogen surface.

Surprisingly, as described herein, Ab bound to a pathogen surface caneffectively trap the pathogen in mucus gel by ensuring at least somelow-affinity bonds to the mucin mesh are present at any given time(illustrated in FIG. 13). For example, virions trapped in CVM cannotreach their target cells, and will instead be shed with post-coitaldischarge and/or inactivated by spontaneous thermal degradation as wellas additional protective factors in mucus, such as defensins (Cole,Curr. Top. Microbiol. Immunol. 306:199 (2006); Doss et al., J. Leukoc.Biol. 87:79 (2010). This discovery provides novel methods for preventingand treating infection, monitoring the effectiveness of vaccines, and/orproviding contraception.

Thus, one aspect of the methods and compositions described herein mayrelate to an isolated antibody comprising an oligosaccharide at aglycosylation site, the oligosaccharide comprising a glycosylationpattern that enhances trapping potency of the antibody in mucus, whereinthe antibody specifically binds an epitope of a target pathogen. Ingeneral, these methods and compositions may include trapping antibodiesdirected against a target viron in mucus (e.g., within the lungs,vagina, etc.).

A further aspect may relate to compositions, e.g., pharmaceuticalcompositions, and kits comprising the antibodies of the invention.

An additional aspect of the invention relates to a method of inhibitingan infection by a pathogen or a disease or disorder caused by aninfection by a pathogen in a subject in need thereof, comprisingadministering to a mucosa of the subject an antibody (e.g., an antibodythat is adapted to have an enhanced trapping potency in mucus) in anamount effective to inhibit an infection.

Another aspect of the invention relates to a method of treating aninfection by a pathogen or a disease or disorder caused by an infectionby a pathogen in a subject in need thereof, comprising administering toa mucosa of the subject the antibody of the invention in an amounteffective to treat the infection.

An additional aspect of the invention relates to a method of monitoringthe effectiveness of a vaccination in a subject that has been vaccinatedagainst a target pathogen, comprising determining in a mucus sample fromsaid subject the amount of an antibody comprising an oligosaccharide ata glycosylation site, the oligosaccharide having a glycosylation patternthat enhances trapping potency of the antibody in mucus, wherein theantibody specifically binds an epitope of said target pathogen.

Typically, viruses must penetrate the mucus of the respiratory tract toreach and infect their target cells in the epithelium. The presentinvention is based in part on the finding that antibodies in therespiratory tract can bind to virions and effectively trap the virionsin mucus gel by ensuring at least some low-affinity bonds to the mucinmesh are present at any given time. Virions trapped in the mucus cannotreach their target cells, and will instead be shed from the respiratorytract with the mucus. This provides novel methods for preventing andtreating infection in the respiratory tract.

Thus, one aspect of the invention relates to method for treating orpreventing a viral infection in a subject in need thereof, the viralinfection characterized by a virion in the lung of the subject, themethod comprising administering, via an inhaled route, an antibody,e.g., a recombinant monoclonal antibody molecule, comprising a human Fcportion and a set of CDRs with specific affinity for the virion, therebytreating or preventing the infection. The antibody may be delivered inan aerosol composition, e.g., by nebulizer.

A further aspect of the invention relates to compositions comprising anantibody, e.g., a recombinant monoclonal antibody molecule, comprising ahuman Fc portion and a set of CDRs with specific affinity for a virionpresent in the lung of a subject, wherein the composition is suitablefor inhalation.

An additional aspect of the invention relates to a kit comprising thecompositions of the invention.

For example, described herein are methods for treating or preventing aninfection by a pathogen (e.g., treating and/or preventing a viralinfection by a virion, treating and/or preventing a bacterial infection,treating and/or preventing a fungal infection) in a subject in needthereof. For example, a method for treating and/or preventing a viralinfection by a virion in a subject in need thereof may include:administering to the subject, via an inhaled route, a recombinantantibody with a specific affinity for the virion, the recombinantantibody comprising a human or humanized Fc region, wherein therecombinant antibody comprises an oligosaccharide having a glycosylationpattern that enhances the trapping potency of the recombinant antibodyin mucus, so that the recombinant antibody binds to the virion to forman antibody/virion complex that is trapped in the subject's mucusthereby treating or preventing the infection.

The administration of the mAb may refer to administering a population ofmAbs that has been enriched for mAbs having a glycosylation pattern thatenhances mucosal trapping.

The recombinant antibody may, when not bound to target (e.g., virion) beweakly interacting with mucus and may therefore freely diffuse throughthe mucus; however, once bound to the target (e.g., virion) to form anantibody/viron complex, it may trap the antibody/virion complex withinthe mucus, allowing it to be removed and/or destroyed by the body. Theconcentration of antibody in the mucus (and particularly theconcentration of mAb having an enhanced trapping efficiency, such ashaving a G0/G0F (i.e. GnGn) glycosylation pattern) may be, e.g., 50ng/mL or greater, (e.g., 100 ng/mL or greater, 200 ng/mL or greater, 500ng/mL or greater, 1 μg/mL or greater, 10 μg/mL or greater, 12.5 μg/mL orgreater, 15.0 μg/mL or greater, 20 μg/mL or greater, etc., e.g., between0.1 μg/mL and 20 μg/mL).

In general, recombinant antibodies having an increased mucosal trappingpotency may comprise an N-linked glycosylation site on the Fc region ofthe antibodies to which the oligosaccharide is attached. For example,the glycosylation pattern may comprise a biantennary core glycanstructure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch. This glycosylation pattern may bewith or without fucose, and/or with or without a bisecting GlcNAc.

In some variations at least 20% (e.g., at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, etc.,and particularly 50% or more, e.g., 55% or greater, 60% or greater, 65%or greater, between 80-100%, etc.) of the recombinant antibodies in thepopulation administered to the patient have a glycosylation patterncomprising the biantennary core glycan structureManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch. For example, at least 50% of therecombinant antibodies in the population have a glycosylation patterncomprising the biantennary core glycan structureManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch. As mentioned, this glycosylationpattern may be with or without fucose, and/or with or without abisecting GlcNAc.

As mentioned, any of these methods may include forming anantibody/virion complex and trapping an antibody/virion complex in themucus. The recombinant antibody may be configured to bind to anon-neutralizing epitope of the virion. Administering may compriseadministering a dose at a sub-neutralization dose level.

Any of these methods may include reducing the mobility of the virion inthe patient's mucus to no more than about 50% relative to its mobilityin water. For example, any of these methods may include reducing thepercentage of virion that can penetrate the patient's mucus by at least10% (e.g., at least 15%, at least 20%, at least 25%, etc.).

The recombinant antibody may be a human or humanized IgG or IgMmonoclonal antibody, or a fragment or derivative thereof.

The recombinant antibody may be formulated as an aerosol composition.For example, the recombinant antibody may be formulated to have aneutral pH (e.g., approximately neutral pH); in some variations therecombinant antibody may be formulated as a hypotonic solution.

Administering may comprise administering a nebulized composition ofrecombinant antibody having a Mass Median Aerodynamic Diameter (MMAD) inthe 2-5 μm range, as measured using a Next Generation Impactor.

Appropriate dosing regimens may include dosing the subject with therecombinant antibody molecule via the inhaled route once every 3 to 24hours. The dosing regimen may include dosing between about 5-100 mg/day(e.g., between about 15-50 mg/day, between about 5-200 mg/day, betweenabout 15-200 mg/day, between about 5-150 mg/day, between about 15-150mg/day, between about 5-100 mg/day, between about 15-100 mg/day, etc.).In some variations the dosing may be configured to maintain a sustainedconcentration in the mucus (e.g., for approximately at least 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 16 hours,20 hours, 24 hours, etc.) of between about 0.5-1000 μg/mL in the mucus(e.g., 1 μg/mL or greater, 10 μg/mL or greater, 15 μg/mL or greater, 20μg/mL or greater, 30 μg/mL or greater, 40 μg/mL or greater, 50 μg/mL orgreater, 60 μg/mL or greater, 70 μg/mL or greater, 80 μg/mL or greater,19 μg/mL or greater, 100 μg/mL or greater, 120 μg/mL or greater, 150μg/mL or greater, etc.). This dosing may be used for adult patientsand/or infant patients. Alternatively, for children (including infants),the dosing may be different.

For example, in some variations dosing may be per body weight. Forexample, for children, the dosing may be in the range of between 0.1-25mg/kg range per day (e.g., 0.5-20 mg/kg per day, 1-15 mg/kg per day,etc.), e.g., 5-75 mg/day for a 5 kg infant. For adults, higher doselevels may be used, e.g., >500 mg/day to even 1 g/day. The concentrationin mucus may be between 0.05-1000 μg/mL (e.g., between about 0.5-1000μg/mL), etc.

As described herein, antibodies of the presently-disclosed subjectmatter (having an enhanced trapping potency in mucus) are capable ofdiffusing through mucus when they are unbound, to allow the antibody tobind a target pathogen or sperm at a desirable rate. However, whenantibodies are bound to the target (e.g., virion), the cumulative effectof the antibody-mucin interactions effectively traps the pathogen orsperm in the mucus. Because in the unbound state the antibodies maydiffuse quickly through the mucus, the half-life of a typical dose islikely to be between 12-24 hours, allowing once or twice daily dosingand still remain above a certain minimal threshold despite the naturalrenewal and clearance of mucus. The unbound antibodies that aretopically delivered (e.g., by inhalation) are not likely to be clearedquickly from the lungs, and the antibody may be at a steady state withthe fluid environment even though the mucin mesh is being clearedquickly.

The patient may be an infant (e.g., a human infant), a child, an adult,or an elderly adult (e.g., 55 or older, 60 or older, 65 or older, 70 orolder, 75 or older, etc.) In some variations the patient isimmunocompromised (e.g., due to a pre-existing condition, due tochemotherapy, due to bone marrow transplant, etc.)

In some embodiments, the virion may be selected from respiratory virusesand/or viruses that are not typically considered respiratory viruses,including but not limited to: Ebola virus, respiratory syncytial virus,influenza virus, adenovirus, human rhinovirus, coronavirus, norovirus,human metapneumovirus, parainfluenza virus, Hantavirus, MERS, Bocavirus,Marburg virus, enterovirus, etc. In particular, the viron may berespiratory syncytial virus.

For example, described herein are methods of immobilizing a respiratoryvirion in mucus of a subject's lung, the method comprising administeringto the subject, via an inhaled route, a recombinant antibody with aspecific affinity for the virion so that the recombinant antibody istrapped in the subject's mucus, the recombinant antibody comprising ahuman or humanized Fc region, wherein the recombinant antibody comprisesan oligosaccharide having a glycosylation pattern that enhances thetrapping potency of the recombinant antibody in mucus, wherein therecombinant antibody is trapped in the subject's mucus at aconcentration of 50 ng/mL or greater (e.g., between 0.1 μg/mL and 20μg/mL).

Also described herein are methods for blocking, preventing oreliminating the proliferation, infiltration or spreading of arespiratory virion in mucus of a subject's lung, the method comprisingimmobilizing the virion by administering to the subject, via an inhaledroute, a recombinant antibody with a specific affinity for the virion sothat the recombinant antibody is trapped in the subject's mucus, therecombinant antibody comprising a human or humanized Fc region, whereinthe recombinant antibody comprises an oligosaccharide having aglycosylation pattern that enhances the trapping potency of therecombinant antibody in mucus.

Also descried herein are nebulized solutions for treating viron,including, in particular, a viron causing a respiratory disorder. Ingeneral, these nebulized solutions may be aerosolized by a nebulizer(e.g., any appropriate nebulizer) to form particles having a mass medianaerodynamic diameter (MMAD) that is less than about 5 μm diameter. Thenebulized solution may generally comprise a recombinant antibody havinga human or humanized Fc region and an oligosaccharide having aglycosylation pattern that enhances the trapping potency of therecombinant antibody in mucus, so that the recombinant antibody binds toa viron to form an antibody/viron complex that is trapped in thesubject's mucus thereby treating or preventing the infection. Thenebulized solution may have a concentration of antibody within theparticles of between 10 mg/mL and 100 mg/mL, and a pH that is neutral orbetween neutral (pH of about 7) and mildly acidic (pH of about 4.5). Insome variations, the nebulized solution may be hypotonic or hypertonicrelative to the lung tissue. Since the compositions descried herein maybe effective outside of the lung tissue (e.g., in the mucus), hypertonic(including mildly hypertonic) solutions may be preferred in somevariations. For example, hypertonic saline may refer to any salinesolution with a concentration of sodium chloride (NaCl) higher thanphysiologic (0.9%). For example, a hypertonic solution may include 2%,3%, 5%, etc. NaCl. In some variations the nebulized solution may bemildly hypertonic (e.g., 2% or 3% NaCl). In some variations, thenebulized solution is isotonic.

In some variations it may be beneficial to include sugars, polyolsand/or amino acids in the nebulized solution. For example in somevariations, the formulation may include one or more (or all) of:histidine, glycine and/or mannitol (e.g., 47 mM Histidine, 4 mM glycineand 5.6% Mannitol). As mentioned, any of these formulations may behypertonic. In some variations the formulation may include NaCl and/orcitrate (e.g., 1.8% NaCl and 10 mM citrate).

Examples of virons may include: Ebola virus, respiratory syncytial virus(RSV), influenza virus, adenovirus, human rhinovirus, coronavirus, humanmetapneumovirus, parainfluenza virus, hantavirus, Middle EasternRespiratory Syndrome (MERS) coronavirus, Bocavirus, and Marburg virus.

In particular, described herein are nebulized solutions for treating orpreventing Respiratory syncytial virus (RSV) in a subject inhaling thenebulized solution, the nebulized solution comprising a recombinantantibody having a human or humanized Fc region and an oligosaccharidehaving a glycosylation pattern that enhances the trapping potency of therecombinant antibody in mucus, so that the recombinant antibody binds tothe RSV to form an antibody/RSV complex that is trapped in the subject'smucus thereby treating or preventing the infection, wherein thenebulized solution comprises particles having a mass median aerodynamicdiameter (MMAD) of between 2-6 μm (e.g., between 3-4.5 μm, less than 6μm, less than 5 μm, etc.) diameter and a concentration of antibodywithin the particles of between 10 mg/mL and 100 mg/mL.

The pH of the nebulized solution may be neutral pH (e.g., pHapproximately 7) or between neutral and mildly acidic (e.g., betweenabout 4.5 and 7.4, between about 4.5 and 7, etc.).

As mentioned, the nebulized solutions described herein may be hypertonic(including mildly hypertonic) relative to the lungs, which may thin themucus, slightly while keeping the antibody within the mucus. In somevariations the nebulized solution may be hypotonic (including mildlyhypotonic) relative to the lungs. In some variations the nebulizedsolution may be isotonic.

In any of these examples, the recombinant antibody may comprise anN-linked glycosylation site on the Fc region of the antibodies to whichthe oligosaccharide is attached. For example, the glycosylation patternmay comprise a biantennary core glycan structure ofManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch. In general, the recombinant antibodymay comprise a human or humanized IgG or IgM monoclonal antibody, or afragment or derivative thereof.

For example, recombinant antibody in the nebulized solution maycomprises palivizumab, or a variant of palivizumab, including but notlimited to motavizumab (or a variant of motavizumab).

Any of the nebulized solutions described herein may include asurfactant. The surfactant may be a nonionic surfactant (e.g.,olyoxyethylene glycol octylphenol ethers: e.g.,C8H17-(C6H4)-(O—C2H4)1-25-OH; Polyoxyethylene glycol alkylphenol ethers:e.g., C9H19-(C6H4)-(O—C2H4)1-25-OH; Polyoxyethylene glycol sorbitanalkyl esters; Sorbitan alkyl esters; block copolymers of polyethyleneglycol and polypropylene glycol, etc.), anionic surfactants (e.g.,dioctyl sodium sulfosuccinate (DOSS); Perfluorooctanesulfonate (PFOS);linear alkylbenzene sulfonates; sodium lauryl ether sulfate;lignosulfonate; sodium stearate, etc.), cationic surfactants (e.g.,benzalkonium chloride (BAC), cetylpyridinium chloride (CPC),benzethonium chloride (BZT), etc.), and zwitterionic surfactants (e.g.,sultaines such as CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), betaines,etc.).

The range of antibody concentration may be particularly critical. Asdescribed above, the concentration of antibody within the particles maybe between 10 mg/mL and 100 mg/mL (e.g., between about 12 mg/mL and 85mg/mL). Although the antibodies may be formed outside of this range ofvalues, the efficacy falls of sharply. For example at concentrationsbetween about 10 mg/mL (e.g., 5 mg/mL), the nebulized solution may haveno appreciable effect, or significantly reduced effect. Concentrationsof antibody greater than about 100 mg/mL were also ineffective, likelydue to aggregation of the mAb, and an increase in the viscosity of thenebulized solution. This unexpected concentration dependence of thenebulized solution may explain, at least in part, the general beliefthat anti-RSV mAbs cannot be aerosolized.

For example, described herein are nebulized solutions for treating orpreventing Respiratory syncytial virus (RSV) in a subject inhaling thenebulized solution, the nebulized solutions comprising a recombinantantibody against an epitope of the F protein of RSV, the recombinantantibody having a human or humanized Fc region and an oligosaccharidehaving a glycosylation pattern that enhances the trapping potency of therecombinant antibody in mucus, so that the recombinant antibody binds tothe RSV to form an antibody/RSV complex that is trapped in the subject'smucus thereby treating or preventing the infection, wherein thenebulized solution comprises particles having a mass median aerodynamicdiameter (MMAD) of between about 2-6 μm (e.g., between about 3-4.5 lessthan about 6 μm, less than about 5 μm diameter, etc.), a pH between 7.0and 8.5, and a concentration of antibody within the particles of between10 mg/mL and 100 mg/mL.

The epitope of the F protein of RSV may be in the A antigenic site, siteII, and/or site Ø, and/or antigenic site VIII, which occupies anintermediate position between the previously defined major antigenicsites II and site Ø.

In general, the methods and compositions (including nebulized solutions)described herein may be directed to respiratory viruses. In addition toRSV, described above, the methods and compositions described herein maybe used for treating one or more of: metapneumovirus, influenza virus,adenovirus, and parainfluenza. For example, the recombinant antibody maybe directed against metapneumovirus, e.g., human mAb 54G10 (J InfectDis. 2015 Jan. 15; 211(2): 216-225, doi: 10.1093/infdis/jiu307),monoclonal antibody (MAb 338, MedImmune, Antiviral Research Volume 88,Issue 1, October 2010, Pages 31-37;https://doi.org/10.1016/j.antiviral.2010.07.001), etc. See also Journalof Virological Methods 254 (2018) 51-64(https://doi.org/10.1016/j.jviromet.2018.01.011), J Virol. 2006 August;80(16): 7799-7806 (doi: 10.1128/JVI.00318-061). J Gen Virol. 2008December; 89(Pt 12): 3113-3118 (doi: 10.1099/vir.0.2008/005199-0), andNat Microbiol. 2017 Jan. 30; 2: 16272 (doi: 10.1038/nmicrobiol.2016.272)for a description of mAbs against metapneumovirus and/or RSV that may beused herein. Another example of two mAbs against metapneumovius aredescribed in U.S. Pat. No. 9,498,531 (note that FIGS. 24A-25B includeone example of the sequences for these two mAbs, referred to in U.S.Pat. No. 9,498,531, as HMB3210 and HMB2430). Any of these mAbs againstmetapneumovirus may be used as described herein, similar to IN-001 andIN-002, described in greater detail, below.

For example, a nebulized solution for treating or preventing arespiratory virus in a subject inhaling the nebulized solution mayinclude: a recombinant antibody having a human or humanized Fc regionand an oligosaccharide having a glycosylation pattern that enhances thetrapping potency of the recombinant antibody in mucus, so that therecombinant antibody binds to the respiratory to form anantibody/respiratory virus complex that is trapped in the subject'smucus thereby treating or preventing the infection, wherein thenebulized solution comprises particles having a mass median aerodynamicdiameter (MMAD) of between about 2 and 6 μm diameter and a concentrationof antibody within the particles of between 10 mg/mL and 100 mg/mL. Asmentioned, the respiratory virus may be one of: Respiratory syncytialvirus (RSV), metapneumovirus, influenza virus, adenovirus, andparainfluenza.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1B show HSV-1 is immobilized in CVM samples with elevatedendogenous anti-HSV-1 IgG but readily mobile in samples with lowendogenous anti-HSV-1 IgG. Fluorescent HSV-1 or control particles wereadded to CVM, and their motions were analyzed by multiple particletracking methods. (A) Representative 20 s traces of HSV-1 (d^(˜)180 nm)and control particles (d^(˜)200 nm) with effective diffusivity (D_(eff))at a time scale r of 1 s within one SEM of the mean. Control particlesinclude muco-inert (PEG-coated; PS-PEG) and muco-adhesive (uncoated; PS)polystyrene beads, which are freely diffusive and trapped in human CVM,respectively. (B) Geometric average D_(eff) (τ=1 s) for PS-PEG, PS andHSV-1 in individual CVM samples from unique donors (n=12, eachexperiment performed independently) as a function of endogenousanti-HSV-1 IgG. Dashed lines represent the D_(eff) cutoff below whichparticles are permanently trapped (moving less than their diameterwithin 1 s). Pearson's correlation coefficients (r) are indicated.

FIGS. 2A-2B show transport characteristics for control beads and HSV-1in CVM. (A) Alpha (a) values for control beads (PS-PEG and PS) and HSV-1in individual CVM samples from unique donors (n=12, each experimentperformed independently) as a function of endogenous anti-HSV-1 IgG. αis the slope of the log-log mean square displacement (<MSD>) vs. timescale plot (α=1 for pure unobstructed Brownian diffusion, e.g.,particles in water, and α becomes smaller as obstruction to particlediffusion increases). Alpha values from all tracking analyses are listedin the table. (B) HSV-1 virions trapped in CVM at short time scalesremain similarly immobilized even at long time scales. HSV-1 in CVMsample with elevated endogenous anti-HSV-1 IgG remain confined to thesame locations over more than 15 min. Time stamps (bottom right corner,magnified at top center; hh:mm:ss) indicate elapsed time in three imagesfrom a representative movie.

FIGS. 3A-3C show HSV-1 immobilization in CVM samples with elevatedendogenous anti-HSV-1 IgG does not correlate with total IgG, IgA or IgMcontent. Geometric average D_(eff) (τ=1 s) for PS-PEG, PS and HSV-1 inindividual CVM samples from unique donors (n=12) as a function of totalendogenous (A) IgG, (B) IgA or (C) IgM. Dashed lines represent theD_(eff) cutoff below which particles are permanently trapped (movingless than their diameter within 1 s).

FIGS. 4A-4B show mobility of HSV-1, but not PS-PEG or PS, is influencedby anti-HSV-1 IgG levels in CVM. (A) HSV-1 is immobilized in native CVMsamples with elevated endogenous anti-HSV-1 IgG, but becomes readilymobile in the same CVM specimens dialyzed to remove ˜90-95% total IgG atconstant sample volume. Control PS-PEG and PS particles remain diffusiveand immobilized, respectively. Dashed line represents the D_(eff) cutoffbelow which particles are permanently trapped (moving less than theirdiameter within 1 s). (B) The addition of exogenous anti-HSV-1 IgG toCVM samples with low levels of endogenous anti-HSV-1 IgG does not alterCVM's diffusional barrier properties to nanoparticles. Control PS-PEGand PS particles remained similarly diffusive and immobilized,respectively, in CVM with low endogenous (“Low endo”), high endogenous(“High endo”) and exogenously added anti-HSV-1 IgG (“Low endo anti-HSV-1IgG”). Differences in average D_(eff) were not statistically significantfor either PS-PEG or PS particles across the three conditions. Dashedline represents the D_(eff) cutoff below which particles are permanentlytrapped (moving less than their diameter within 1 s).

FIGS. 5A-5C show anti-HSV-1 polyclonal human IgG added to CVM sampleswith low endogenous anti-HSV-1 IgG potently traps HSV-1. HSV-1 mobilitywas quantified in aliquots of the same CVM samples with differentamounts of anti-HSV-1 IgG added. (A) Comparison of effective diffusivity(D_(eff); τ=1 s) for HSV-1 in CVM samples (n=7, each experimentperformed independently) with different amounts of total anti-HSV-1 IgG(sum of endogenous and added IgG). Different colored circles representdistinct samples. (B) In vitro neutralization vs. trapping potency ofanti-HSV-1 IgG. Neutralization was assayed based on reduction of HSVplaque formation in Vero cells; trapping was defined as D_(eff) (L=1 s)below 0.01 μm²/s. Total IgG was averaged across samples for eachtreatment group. Error bars represent SEM. * indicates statisticallysignificant difference compared to respective controls (p<0.05). (C)Distribution of particle speeds in samples treated with 1 μg/mL IgG(annulus chart), and estimated concentration of total IgG (μg/mL) neededfor 50% trapping (number in center). Donor ID is indicated for eachsample, with colors matching those in (A).

FIGS. 6A-6B show trapping HSV virions with polyclonal anti-HSV-1 andmonoclonal anti-gD human IgG based on weak IgG-mucus interactions. (A)HSV-1 mobility in native CVM samples with low endogenous anti-HSV-1 IgGtreated with 1 μg/mL polyclonal anti-HSV-1 IgG (“Anti-HSV-1 pAb”),monoclonal anti-gD IgG produced in 293 cells (“Anti-gD mAb”) oranti-biotin monoclonal IgG produced in 293 cells (“Control”). Dashedline represents the D_(eff) cutoff below which particles are permanentlytrapped (moving less than their diameter within 1 s). * indicatesstatistically significant difference compared to the Control group(p<0.01); the difference between the Anti-HSV-1 pAb and Anti-gD mAbgroups was not statistically significant. (B) Ratio of diffusioncoefficients in CVM vs. saline (D_(muc)/D_(sal)) for different Abmeasured by fluorescence recovery after photobleaching (FRAP). Forcomparison, FITC-labeled human IgG (“Control”) is included. Shadedregions indicate the range of values (dark grey=averages and lightgrey=average±standard deviation, respectively).

FIGS. 7A-7C show IgG-mucus interaction is Fe- andglycosylation-dependent. (A) Preparation of anti-HSV-1 F(ab′)₂ confirmedby SDS-PAGE, (B) Preparation of deglycosylated anti-HSV-1 IgG confirmedby lectin-binding assay (absorbance of IgG-bound ConA normalized toamount of IgG). Error bars represent SEM. (C) Mobility (D_(eff); τ=1 s)of HSV-1 in CVM with low endogenous anti-HSV-1 IgG incubated withvarious HSV-1 specific Ab: 1 μg/mL affinity-purified native IgG(“Intact”), 667 ng/mL F(ab′)₂, and 1 deglycosylated IgG compared toHSV-1 in native CVM (“None”). Distinct samples (n=4-5, each experimentperformed independently) are indicated with different color circles;averages are indicated by solid lines. Dashed line represents theD_(eff) cutoff below which particles are permanently trapped (movingless than their diameter within 1 s). * indicates statisticallysignificant difference (p<0.05).

FIGS. 8A-8C show a non-neutralizing monoclonal IgG₁ against the gGepitope protects against vaginal HSV-2 infection in mice, withoutdecreasing the extent of infection in mice that became infected. (A) Invitro neutralization of (% Neut.) vs. (B) in vivo protection againstHSV-2. Neutralization was assayed based on reduction of HSV plaqueformation in Vero cells. Depo-Provera®-treated mice were inoculated withHSV-2 mixed with control or anti-gG IgG. Infection was assayed threedays post-inoculation by detection of virus in vaginal lavages using theELVIS®HSV Test System. (C) Degree of HSV-2 shedding detected in vaginallavages of all mice (filled circles), or of infected mice only (opencircles). In vivo protection by 33 μg/mL anti-gG IgG is lost when mouseCVM is removed by gentle washing using a syringe pump. Data representfour independent experiments, each with n=10 mice per group. Error barsrepresent SEM. * indicates statistically significant difference comparedto control (p<0.05).

FIGS. 9A-9C show protection by anti-gG IgG₁ is lost when mouse CVM isremoved by gentle vaginal wash. (A) Mucin concentration in vaginal fluidcollected from the native or gently washed mouse vagina, YOYO-1 stainingfor vaginal epithelial cell damage in gently washed or conventionallylavaged and swabbed (cotton tip) mice, and H&E stained transversesections of native vs. gently washed mouse vaginal tissue, N.D.=no data.(B) % infected among mice treated with 33 μg/mL anti-gG IgG with orwithout gentle washing to remove vaginal mucus. (C) The gentle wash didnot alter the extent of infection in infected mice. Data represent atleast three independent experiments, each with n−10 mice per group(total n=30-40 per group). Error bars represent SEM, * indicatesstatistically significant difference compared to control (p<0.05).

FIGS. 10A-10B show mobility of individual Salmonella typhimuriumbacteria in control or anti-LPS monoclonal IgG-treated mousegastrointestinal mucus. (A) Microscopy setup that enables measurement ofbacterial mobility in real time directly in mucus overlaying intestinalepithelial tissue excised from mice. The epithelium was NOT subjected towashing and hence the physiological mucus layer is intact. (B) Thefraction of motile Salmonella typhimurium and their velocities in mucusoverlaying different parts of the GI tract in native vs. anti-LPS IgG1treatment. Similar findings were observed with anti-flagella Ab as wellas with IgG2a.

FIGS. 11A-11B show mobility of individual Salmonella typhimuriumbacteria in control or anti-LPS monoclonal IgG-treated mousegastrointestinal mucus. (A) Fraction moving and (B) average velocity.Note that average velocity for the anti-LPS IgG group is 0.036 μm/s,85-fold lower than the average velocity for the control group.

FIG. 12 shows the role of N-glycans in IgG-mucin interactions. In theoligosaccharide structure, the square is N-acetylglucosamine (GlcNAc),the triangle is fucose, the diamond is sialic acid (Neu5Gc), the lightgray circle is galactose (Gal), and the dark gray circle is mannose.

FIG. 13 shows the proposed mechanism of Ab-mediated trapping of virusesin mucus. Schematic showing (panel a) HSV readily penetrates native CVMwith little to no endogenous anti-HSV IgG, and (panel b) anti-HSV IgGtraps HSV in CVM by multiple transient, low affinity bonds with mucins.By forming only short-lived, low-affinity bonds with mucus, free Ab,such as IgG, are able to diffuse rapidly through mucus and binds toviruses. As IgG molecules accumulate on the virus surface, they formmultiple low-affinity bonds between the virus and mucus gel. Asufficient number of these transient low-affinity bonds ensure virusesare effectively trapped in mucus at any given time, thereby reducing theflux of infectious virions that can reach target cells. Arrows indicatethe small fraction of free (not virus-bound) IgG (˜10-20%) that willinteract with mucins at any moment in time.

FIGS. 14A-14B show characterization of Ebola virus-like particles (VLP).(A) Incorporation of Ebola glycoprotein (GP) confirmed via Western blot.Ebola VLP were prepared by transfecting 293T cells with expressionplasmids encoding Ebola GP and HIV Gag-mCherry. Null VLP were preparedfrom cells transfected with HIV Gag-mCherry only. rGPd™ (recombinantEbola GP) serves as a positive control for the 110 kd GP band. (B) EbolaVLP immunoprecipitated with ZMapp™ or individual Ebola-binding IgG(c2G4, c116C6FR1 and c4G7). The Ebola-binding mAbs were modified to havea glycosylation pattern having a higher than usual G0 glycosylationpattern, as described herein, to enhance mucosal binding. The G0 contentof the antibodies used herein was 80% or more (e.g., between 85-95%).rGPd™ serves as a positive control for the GP band, while Null VLP andα-Biotin serve as negative controls confirming the specificity of ZMapp™binding to Ebola VLP.

FIGS. 15A-15D show diffusion in human airway mucus (AM) of Ebola VLP andcomparably sized polystyrene nanoparticles that are either carboxylatedand mucoadhesive (PS-COOH) or modified with polyethylene glycol andmuco-inert (PS-PEG). (A) Representative trajectories for VLP andparticles exhibiting effective diffusivities within one SEM of theensemble average at a time scale of 0.2667 s. (B) Distributions of thelogarithms of individual particle effective diffusivities (D_(eff)) at atime scale of 0.2667 s. Log D_(eff) values to the left of the dashedline correspond to particles with displacements of less thanapproximately 200 nm (i.e., roughly twice the particle diameter) within0.2667 s or D_(eff) approximately 20-fold reduced from theoreticaldiffusivity in water. (C) Ensemble-averaged geometric mean squaredisplacements (<MSD>) as a function of time scale. (D) Ensemblegeometric D_(eff) at a timescale of 0.2667 s and fraction of mobileparticles are plotted for distinct samples with averages indicated bysolid lines. Data represent the ensemble average of 8 independent AMspecimens. Error bars represent standard error of the mean (SEM). *indicates a statistically significant difference compared to PS-COOH(p<0.05) based on a one-tailed, paired Student's t-test.

FIGS. 16A-16C show diffusion of Ebola VLP in human AM that is untreated(No Ab) or treated with either ZMapp™ or individual Ebola-binding IgG(c2G4, c116C6FR1, c4G7). (A) Representative trajectories for VLPexhibiting effective diffusivities within one SEM of the ensembleaverage at a time scale of 0.2667 s. (B) Ensemble-averaged geometricmean square displacements (<MSD>) as a function of time scale. (C)Distributions of the logarithms of individual particle effectivediffusivities (D_(eff)) at a time scale of 0.2667 s. Log D_(eff) valuesto the left of the dashed line correspond to particles withdisplacements of less than approximately 200 nm (i.e., roughly twice theparticle diameter) within 0.2667 s or D_(eff) approximately 20-foldreduced from theoretical diffusivity in water. Data represent theensemble average of 8-9 independent AM specimens. Error bars representstandard error of the mean (SEM). * indicates a statisticallysignificant difference compared to No Ab (p<0.05) based on a one-tailed,paired Student's t-test.

FIGS. 17A-17B show diffusion and first passage time of Ebola VLP inhuman AM that is untreated (No Ab) or treated with either ZMapp™ orindividual Ebola-binding IgG (c2G4, c116C6FR1, c4G7). (A) Ensemblegeometric average D_(eff) at a timescale of 0.2667 s and fraction ofmobile particles. (B) Estimated time for 10% and 50% of VLP andparticles to diffuse through a 50 μm thick mucus layer. Data representthe ensemble average of 8-9 independent AM specimens. Error barsrepresent standard error of the mean (SEM). * indicates a statisticallysignificant difference compared to No Ab (p<0.05) based on a one-tailed,paired Student's t-test.

FIGS. 18A-18E show (e.g., FIGS. 18A-18D) representative transverse 50 μmthick frozen tissue sections showing the distribution of Ebola VLP inthe mouse trachea treated with (A, B) PBS or (C, D) ZMapp™. Redcorresponds to Ebola VLP, and blue corresponds to DAPI-stained cellnuclei. Arrows indicate the inner lining of the trachea. (E)Quantification of Ebola VLP signal in mouse trachea treated with PBS(“Control”) or ZMapp™ (“ZMapp-Treated”) compared to blank tissue(“Blank”). Data represent n=3 mice per group with on average 10 tissuesections quantified per mouse. Error bars represent standard error ofthe mean (SEM). * indicates a statistically significant difference(p<0.05) based on a two-tailed Student's t-test assuming unequalvariance.

FIGS. 19A-19B show diffusion of RSV in human AM. (A) Representativetraces of mobile vs. trapped RSV. (B) Distributions of the logarithms ofindividual particle effective diffusivities (Deft).

FIGS. 20A-20B show (A) cryosections of mouse airways inoculated withfluorescent RSV (red) treated with either PBS or RespiraClear, anaerosol formulation of mAb against RSV that is a “muco-trapping”antibody. In FIGS. 20A-20B, the RespiraClear is a muco-trapping form ofpalivizumab, as described herein. For example, RespiraClear in thisexample is a mAb having the heavy and light chain variable regions ofpalivizumab and a glycosylation pattern having a biantennary core glycanstructure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch. Cells are stained with DAPI. Thevirus is retained in the airways treated with PBS (left columns) but iscleared from the airways in mouse treated with 1 μg/mL RespiraClear(right columns) Both virus and treatments are dosed at 25 μL using anaerosolizer (e.g., a PennCentury microsprayer), and mouse is sacrificed30 mins after treatment. (B) Quantitative analysis of images fromhistology studies. Studies were performed on n=6 mice with at least 10sections analyzed per mice.

FIG. 21 shows the reduction in infectious viral titers in the cotton ratlung after intranasal administration of antibody.

FIGS. 22A-22B show (A) photographs of a next generation impactor (NGI)for size verification of aerosol particles, following Privigendeposition. The right photograph is a zoomed in image of stage three ofthe NGI. (B) Inertial impaction results of nebulized Privigen whenoperating a NGI at 15 L/min for three minutes. Error bars represent onestandard deviation above and below the mean (n=3).

FIG. 23A-23C illustrates aerosol characteristics and mAb stabilityfollowing nebulization using a vibrating mesh nebulizer for a solutionof 15 mg/mL of IN-002 (a glycosylated population of motavizumab). FIG.23A illustrates the aerodynamic particle size distribution for aerosolsgenerated from the solution of 15 mg/mL of IN-002, measured using a nextgeneration impactor (NGI). FIG. 77B shows human IgG ELISA for nebulizedIN-002 collected from different stages of NGI, compared to unnebulizedIN-002. FIG. 23C illustrates the results of whole RSV-A2 coated ELISAfor nebulized IN-002 collected from different stages of NGI, compared tounnebulized IN-002. In FIGS. 23B and 23C, a value of ˜100% indicatesthat the ELISA′ assay detect no difference in the structure and antigenaffinity of nebulized IN-002 compared to IN-002 that has not beennebulized.

FIGS. 23D-23F illustrate aerosol characteristics and mAb stabilityfollowing nebulization using vibrating mesh nebulizer for a solution of5.4 mg/mL IN-002. FIG. 23D shows aerodynamic particle size distributionfor aerosols generated from the solution of 5.4 mg/mL IN-002, measuredusing a next generation impactor (NGI). FIG. 23E shows human IgG ELISAfor nebulized IN-002 collected from different stages of NGI, compared tounnebulized IN-002. FIG. 23F shows whole RSV-A2 coated ELISA fornebulized IN-002 collected from different stages of NGI, compared tounnebulized IN-002. In FIGS. 23E and 23F, a value of ˜400% indicatesthat the ELISA assay detect no difference in the structure and antigenaffinity of nebulized IN-002 compared to IN-002 that has not beennebulized.

FIGS. 24A-E show lung pathology in RSV-infected lambs. Lambs weretreated daily with either saline or two different formulations ofmuco-trapping mAb (IN-001, a glycosylated population of palivizumab, andIN-002, a glycosylated population of motavizumab) against RSV, beginningon Day 3 unless otherwise indicated. The images were taken at necropsyon Day 6 post-infection. FIG. 24A shows un-infected tissue (negativecontrol) and FIG. 24B shows infected, saline treated tissue (positivecontrol). FIG. 24C shows tissue from an infected lamb treated withIN-001. FIG. 24D shows tissue from an infected lamb treated with IN-002.FIG. 24E shows tissue from an infected lamb treated with IN-002 on day2.

FIG. 24F is a chart showing gross lesion scores, quantified by percentof lung tissue involved or have RSV lesions, for the different animals *indicates p<0.05; ** indicates p<0.005.

FIG. 25 is a chart showing infectious fluorescent focus-forming units(FFU) per gram of lung homogenates for neonatal lambs infected with RSVon day 0, which received daily treatment either starting on day 2 or day3 post-infection, as indicated. The lambs were sacrificed on day 6.Values below the axis break represent linear rather than logarithmicvalues (i.e. value of zero represents 0 FFU/g). * indicates p<0.05.

FIG. 26 is a chart showing infectious fluorescent focus-forming units(FFU) per mL of bronchial alveolar lavage fluid (BALF). In this example,neonatal lambs were infected with on day 0, and received daily treatmenteither starting on day 2 or day 3 post-infection, as indicated. Thelambs were sacrificed on day 6, which is when BALF was collected. Valuesbelow the axis break represent linear rather than logarithmic values(i.e. value of zero represents 0 FFU/mL). * indicates p<0.05.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery andcharacterization of weak binding interactions between antibodies andmucins and the ability of such antibodies to stop the penetration ofpathogens through mucus layers. The antibody-mucin interaction can beused advantageously in methods for preventing and treating infection,providing contraception, and monitoring the effectiveness of vaccines.

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for production of recombinant mid syntheticpolypeptides, antibodies or antigen-binding fragments thereof,manipulation of nucleic acid sequences, and production of transformedcells. Such techniques are known to those skilled in the art. See, e.g.,SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (ColdSpring Harbor, N.Y., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley &Sons, Inc., New York).

All publications, patent applications, patents, nucleotide sequences,amino acid sequences and other references mentioned herein areincorporated by reference in their entirety.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units is also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The transitional phrase “consisting essentially of” means that the scopeof a claim is to be interpreted to encompass the specified materials orsteps recited in the claim, “and those that do not materially affect thebasic and novel characteristic(s)” of the claimed invention. See, In reHerz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis inthe original); see also MPEP § 2111.03.

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotidebases, and may be RNA, DNA or DNA-RNA hybrid sequences (including bothnaturally occurring and non-naturally occurring nucleotide), but ispreferably either single or double stranded DNA sequences.

As used herein, an “isolated” antibody means an antibody separated orsubstantially free from at least some of the other components of thenaturally occurring organism or virus, for example, the cell structuralcomponents or other polypeptides or nucleic acids commonly foundassociated with the antibody. The term also encompasses antibodies thathave been prepared synthetically.

By the terms “treat,” “treating,” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is a delay in the progressionof the condition.

As used herein, the terms “prevent,” “prevents,” or “prevention” and“inhibit,” “inhibits,” or “inhibition” (and grammatical equivalentsthereof) are not meant to imply complete abolition of disease andencompasses any type of prophylactic treatment that reduces theincidence of the condition, delays the onset of the condition, and/orreduces the symptoms associated with the condition after onset.

An “effective,” “prophylactically effective,” or “therapeuticallyeffective” amount as used herein is an amount that is sufficient toprovide some improvement or benefit to the subject. Alternativelystated, an “effective,” “prophylactically effective,” or“therapeutically effective” amount is an amount that will provide somedelay, alleviation, mitigation, or decrease in at least one clinicalsymptom in the subject. Those skilled in the art will appreciate thatthe effects need not be complete or curative, as long as some benefit isprovided to the subject.

As used herein, the term “trapping potency” refers to the ability of anantibody that specially binds to a target pathogen or sperm to inhibitmovement of the pathogen or sperm through mucus. Trapping potency can bemeasured by methods known in the art and as disclosed herein. Trappingpotency can be quantitated, e.g., as the amount of antibody (e.g.,concentration of antibody in mucus) needed to reduce the mobility of atleast 50% of the pathogen or sperm Within the mucus gel to at leastone-tenth of its mobility in solution (e.g., saline). Mobility in mucuscan be measured using techniques well known in the art and describedherein. Alternatively, trapping potency can be quantitated as thereduction in percentage of pathogens or sperm that penetrate mucus.

The term “enhances trapping potency” refers to an antibody comprising anoligosaccharide that provides an increased trapping potency relative tothe trapping potency of antibodies as found in nature and/or antibodiesprior to any modification and/or selection.

As used herein, the term “bind specifically” or “specifically binds” inreference to an antibody of the presently-disclosed subject matter meansthat the antibody of the invention will bind with an epitope (includingone or more epitopes) of a target pathogen or sperm, but does notsubstantially bind to other unrelated epitopes or molecules. In certainembodiments, the term refers to an antibody that exhibits at least about60% binding, e.g., at least about 70%, 80%, 90%, or 95% binding, to thetarget epitope relative to binding to other unrelated epitopes ormolecules.

Antibodies and Compositions

The presently-disclosed subject matter includes antibodies,compositions, and methods for inhibiting and/or treating pathogeninfection, eliminating pathogen from a mucosal surface, providingcontraception, and/or monitoring the effectiveness of vaccines. Inparticular, the presently-disclosed subject matter relates to antibodiesand compositions capable of trapping pathogens and sperm in mucus,thereby inhibiting transport of pathogens or sperm across or throughmucus secretions.

The prevailing view of how antibodies protect a subject at mucosalsurfaces assumes that neutralization of the pathogen is the primarymechanism of protection. Surprisingly and unexpectedly, in light of thiswidespread view, the present inventors disclose herein thatneutralization is not necessary to protect against infection at mucosalsurfaces in a subject. Indeed, it is demonstrated herein thatsub-neutralization doses of antibodies to neutralizing epitopes ofpathogens can be quite effective at inhibiting infection. Furthermore,it is demonstrated herein that use of antibodies to non-neutralizingepitopes of pathogens can also be quite effective at inhibitinginfection.

Antibodies are naturally found in mucus. The current thoughts onantibody-mediated mucosal protection are that secretory IgA (sIgA)antibodies are important for protection because very large amounts ofthis isotype are found in the gastrointestinal tract. It is furtherthought that IgG does not play a role in mucosal protection. However,IgG is the dominant isotype in genital secretions and there aresubstantial quantities of IgG found in respiratory mucus secretions. Incontrast to the prevailing thought in the scientific community, it isshown herein that certain antibodies, e.g., IgG, found in mucus, e.g.,CVM, can diffuse rapidly through the mucus, slowed only slightly byweak, transient adhesive interactions with mucins within the mucus. Thisrapid diffusion allows antibodies to accumulate rapidly on pathogen orsperm surfaces. When a plurality of antibodies have accumulated on thesurface of a pathogen, the adhesive interactions between the pluralityof antibodies and the mucus become sufficient to trap the bound pathogenor sperm in the mucus, thereby preventing infection/providingcontraception. Pathogens or sperm trapped in CVM cannot reach theirtarget cells in the mucosal surface, and will instead be shed withpost-coital discharge and/or inactivated by spontaneous thermaldegradation as well as additional protective factors in mucus, such asdefensins (Cole, Curr. Top. Microbiol. Immunol. 306:199 (2006); Doss etal., J. Leukoc. Biol. 87:79 (2010). As disclosed herein, this pathogentrapping activity provides for protection without neutralization, andcan effectively inhibit infection at sub-neutralization doses and/orusing antibodies to non-neutralizing epitopes of a pathogen.

The present invention additionally provides the discovery that thelow-affinity interactions that an antibody forms with mucins are notonly Fe-dependent, but also influenced by antibody glycosylation.

Accordingly, the presently-disclosed subject matter includes an isolatedantibody comprising an oligosaccharide at a glycosylation site, theoligosaccharide comprising, consisting essentially of, or consisting ofa pattern correlating with (providing) enhanced trapping potency of theantibody in mucus, and wherein the antibody specifically binds anepitope of a target pathogen or sperm. The unique glycosylationpattern/unique oligosaccharide component of the antibody is designed tomaximize trapping potency of the antibody once a plurality of antibodiesare bound to the target pathogen or sperm, without unduly hindering theability of the unbound antibody to diffuse readily through mucus torapidly bind a target pathogen or sperm. In certain embodiments, theantibody is one that exhibits a mobility in mucus that is reduced nomore than about 50%, e.g., no more than about 40%, 30, 20%, 10%, or 5%,relative to its mobility in solution (e.g., saline or water) andeffectively traps a target pathogen or sperm in mucus based on aplurality of bound antibodies (e.g., at least 50% of pathogens or spermslowed by at least 90%). In some embodiments, the antibody reduces themobility of at least 50%, e.g., at least 60%, 70%, 80%, or 90% or moreof the pathogen or sperm by at least 90%, e.g., at least 95%, 96%, 97%,98%, or 99% or more. In other embodiments, the antibody reduces thepercentage of pathogens or sperm that can penetrate mucus by at least10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.Based on the disclosure herein, one of skill in the art can readilyidentify/design oligosaccharide patterns that provide the desiredtrapping potency. In other embodiments, the antibody has a sufficientbinding rate to an epitope of the target pathogen or sperm to accumulateon the surface of the target pathogen or sperm at sufficient levels totrap the target pathogen or sperm in mucus within one hour (e.g., within30 minutes or 15 minutes) at an antibody concentration in the mucus ofless than 5 μg/mL (e.g., less than 1 μg/mL or 0.1 μg/mL).

In some embodiments, the oligosaccharide component is bound to anN-linked glycosylation site in an Fc region of the antibody. TheN-linked glycosylation site can be an asparagine residue on the Fcregion of the antibody, for example, the Asn 297 asparagine residue. Theamino acid numbering is with respect to the standard amino acidstructure of a human IgG molecule.

The N-glycan structure on human IgG-Fc is typically dominated by abiantennary core structure that shares a common core sugar sequence (asshown in FIG. 12),Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser/Thr, with “antennae”initiated by N-acetylglucosaminyltransferases (GlcNAcTs) that helpattach additional sugars to the core. In IgG found in human serum, themost common structures are those that contain both N-acetylglucosamineon each branch with one terminal galactose (39%), two terminal galactose(20%), or one terminal galactose and one terminal sialic acid (15%).Together, antibodies that comprise at least one terminal galactoserepresents about 74% of the IgG-Fc glycoforms. A pure GnGn form (withterminal N-acetylglucosamine on each branch without terminal galactoseor sialic acid) represents about 26% of the IgG-Fc glycoforms.

In some embodiments, the oligosaccharide component, i.e., the glycan,attached to the antibody comprises, consists, essentially of, orconsists of the core structure depicted in FIG. 12. In some embodiments,the glycan attached to the antibody comprises, consists essentially of,or consists of the core structure depicted in FIG. 12 minus the fucoseresidue. In some embodiments, the glycan comprises, consists essentiallyof, or consists of the full structure depicted in FIG. 12. In otherembodiments, the glycan does not contain any galactose residues. Incertain embodiments, the glycan comprises the core structure as depictedin FIG. 12 and additional saccharide residues that do not includegalactose. Without being limited by theory, it is believed that thepresence of galactose compromises trapping potency. Antibodies withglycoforms that do not contain galactose represent just a small fractionof the entire repertoire of glycoforms found in nature. The use of apopulation of antibodies enriched with desirable glycoforms (whethernaturally occurring or modified glycans) is advantageously used fortrapping pathogens and sperm in mucus.

In some embodiments, the antibody of the invention is a mixture ofantibodies having different oligosaccharide components. In someembodiments, the mixture of antibodies comprises at least about 30%antibodies having the core glycan structure depicted in FIG. 12 with orwithout the fucose residue, e.g., at least about 40%, 50%, 60%, 70%,80%, 90% or more. In other embodiments, the mixture of antibodiescomprises at least about 5% antibodies having the full glycan structuredepicted in FIG. 12, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or more. In some embodiments, the mixture of antibodiescomprises at least about 30% antibodies having the core glycan structuredepicted in FIG. 12, e.g., at least about 40%, 50%, 60%, 70%, 80%, 90%or more and at least about 5% antibodies having the full glycanstructure depicted in FIG. 12, e.g., at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more.

In some embodiments, the mixture of antibodies is the mixture generatedin a human cell line, e.g., a 293 cell line, e.g., a 293T cell line.

The antibody is useful for binding target pathogens to trap the pathogenin mucus to inhibit infection by the pathogen. Target pathogens of theantibody can include any pathogen that can infect a subject through amucus membrane. Pathogens can be in the categories of algae, bacteria,fungi, parasites (helminths, protozoa), viruses, and subviral agents.Target pathogens further include synthetic systems comprising an antigenhaving an epitope, for example particles or particulates (e.g.,polystyrene beads) comprising attached proteins, e.g., as might be usedfor bioterrorism.

Pathogens include those that cause sexually-transmitted diseases (listedwith the diseases caused by such pathogens), including, withoutlimitation, Neisseria gonorrhoeae (gonorrhea); Chlamydia trachomatis(chlamydia, lymphogranuloma venereum); Treponema pallidum (syphilis);Haemophilus ducreyi (chancroid); Klebsiella granulomatis orCalymmatobacterium granulomatis (donovanosis) Mycoplasma genitalium,Ureaplasma urealyticum (mycoplasmas); human immunodeficiency virus HIV-1and HIV-2 (HIV, AIDS); HTLV-1 (T-lymphotrophic virus type 1); herpessimplex virus type 1 and type 2 (HSV-1 and HSV-2); Epstein-Barr virus;cytomegalovirus; human herpesvirus 6; varicella-zoster virus; humanpapillomaviruses (genital warts); hepatitis A virus, hepatitis B virus,hepatitis C virus (viral hepatitis); molluscum contagiosum virus (MCV);Trichomona vaginalis (trichomoniasis); and yeasts, such as Candidaalbicans (vulvovaginal candidiasis). The antibodies and compositions mayalso be active against other diseases that are transmitted by contactwith bodily fluids that may also be transmissible by sexual contact andare capable of being prevented by administration of the compositionsaccording to this invention. Accordingly, the phrase, “sexuallytransmitted diseases (STDs),” is to be interpreted herein as includingany disease that is capable of being transmitted in the course of sexualcontact, whether or not the genital organs are the site of the resultingpathology.

Pathogens also include those that cause respiratory diseases, including,without limitation, influenza (including influenza A, B, and C); severeacute respiratory syndrome (SARS); respiratory syncytial virus (RSV);parainfluenza; adenovirus; human rhinovirus; coronavirus; and norovirus.

Other pathogens include, without limitation, Salmonella and Escherichiacoli.

Pathogens include those that affect non-human animals, such aslivestock, e.g., swine (e.g., porcine epidemic diarrhea virus (PEDV),transmissible gastroenteritis virus (TGEV), rotavirus, classical swinefever virus (CSFV), porcine circovirus type 2 (PCV2),encephalomyocarditis virus (EMCV), porcine reproductive and respiratorysyndrome virus (PRRSV), porcine parvovirus (PPV), pseudorabies virus(PRV), Japanese encephalitis virus (JEV), Brucella, Leptospira,Salmonella, and Lawsonia intracellularis, Pasteurella multocida,Brachyspira hyodysenteriae, Mycoplasma hyopneumoniae), ruminants (e.g.,bovine virus diarrhoea virus (BVDV), border disease virus (BDV), bovinepapular stomatitis virus (BPSV), pseudocowpox virus (PCPV), Pasteurellahaemolytica, Pasteurella multocida, Haemophilus somnus, Haemophilusagnii, Moraxella bovis, Mycoplasma mycoides, Theileria annulata,Mycobacterium avium paratuberculosis), ungulates (e.g., Brucellaabortus, Mycobacterium bovis, Theileria parva, Rift Valley fever virus,foot-and-mouth disease virus, lumpy skin disease virus), horses (e.g.,Rhodococcus equi, Salmonella choleraesuis, Pasteurella multocida, equineherpesvirus-1, equine herpesvirus-4, equine influenza virus,Streptococcus equi), poultry (e.g., fowl pox virus, Newcastle diseasevirus, Marek's disease virus, avian influenza virus, infectious bursaldisease virus (IBDV), avian infectious bronchitis virus (IBV)), and thelike.

The terms virus and viral pathogen are used interchangeably herein, andfurther refer to various strains of virus, e.g., influenza is inclusiveof new strains of influenza, which would be readily identifiable to oneof skill in the art. The terms bacterium, bacteria, and bacterialpathogen are used interchangeably herein, and further refer toantibiotic-resistant or multidrug resistant strains of bacterialpathogens. As used herein when referring to a bacterial pathogen, theterm “antibiotic-resistant strain” or “multidrug resistant strain”refers to a bacterial pathogen that is capable of withstanding an effectof an antibiotic or drug used in the art to treat the bacterial pathogen(i.e., a non-resistant strain of the bacterial pathogen).

In some embodiments, it is contemplated that an antibody according tothe presently-disclosed subject matter is capable of broadly binding toviruses containing lipid envelopes, which are not necessarily specificto one virus.

As noted above, it was surprisingly discovered that sub-neutralizationdoses of an antibody can be used to effectively trap a target pathogenor sperm in mucus. As such, in some embodiments, wherein the antibodyspecifically binds a neutralizing epitope of the target pathogen, asub-neutralization dose can be used. A sub-neutralization doses is adose below that which would be needed to achieve effectiveneutralization. For example, in the case of polyclonal anti-HSV gGantibodies targeting HSV, as described hereinbelow, an effectiveneutralization dose is approximately 5 μg/mL. However, effectivetrapping using the antibody can be achieved at a dose below 5 μg/mL, andeven below a dose of 1 μg/mL.

As will be recognized by one of skill in the art, doses appropriate fortrapping bacterial pathogens can be higher in some embodiments than thedoses appropriate for trapping viral pathogens. It will further berecognized that appropriate doses may differ between pathogens, betweenmucosal surfaces, and also between individuals. It will also berecognized that different subjects and different mucosal surfaces mayhave different optimal glycan patterns and optimal antibody-mucinaffinities, contributing to different optimal doses.

It is further proposed herein that antibodies that selectively bindnon-neutralizing epitopes of a target pathogen can be used toeffectively trap the target pathogen in mucus. As such, in someembodiments, the antibody specifically binds a non-neutralizing epitope,e.g., one or more non-neutralizing epitopes.

The presently-disclosed subject matter further includes an antibody thatselectively binds a conserved epitope of a target pathogen, a benefit oftargeting a conserved epitope would be to preserve efficacy of theantibody as against new strains of the pathogen. Targeting such epitopeshas been avoided at times in the past because they were viewed as beingineffective targets; however, in view of the disclosure herein thatnon-neutralizing epitopes can serve as effective targets and/or thatsub-neutralization doses can be effective for inhibiting infection,previously dismissed conserved epitopes of target pathogens can be seenas effective targets.

Antibodies of the invention are useful for binding sperm to trap thesperm in mucus to inhibit fertilization of an egg by the sperm. Spermspecific antigens that can be used as antibody targets are well known inthe art. See, e.g., U.S. Pat. Nos. 8,211,666, 8,137,918, 8,110,668,8,012,932, 7,339,029, 7,230,073, and 7,125,550, each incorporated byreference in its entirety.

As noted above, it was determined that the low-affinity bindinginteractions that an antibody forms with mucins are influenced byantibody glycosylation, and are also Fc-dependent. As such, thepresently-disclosed subject matter includes antibodies having apreserved and/or engineered. Fc region. Such antibodies can be, forexample, one or more of IgG, IgA, IgM, IgD, or IgE. In certainembodiments, the antibodies are IgG. In some embodiments, the antibodiesare one or more subclasses of IgG, e.g., IgG₁, IgG₂, IgG₃, IgG₄, or anycombination thereof.

In some embodiments, the antibody has a sufficient binding rate and/orbinding affinity to an epitope of the target pathogen or sperm toaccumulate on the surface of the pathogen or sperm at sufficient levelsto trap the pathogen or sperm within one hour after administration ofthe antibody at an antibody concentration of less than about 5 μg/mL.The term “trap” in this instance refers to reduction of further movementthrough the mucus. In some embodiments, the target pathogen or sperm istrapped within about 30 minutes, e.g., about 25, 20, 15, or 10 minutesafter administration of the antibody. In some embodiments, the antibodytraps the target pathogen or sperm at an antibody concentration of lessthan about 4, 3, 2, or 1 μg/mL.

The following discussion is presented as a general overview of thetechniques available for the production of antibodies; however, one ofskill in the art will recognize that many variations upon the followingmethods are known.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep, camel,or human, or can be a chimeric or humanized antibody. See, e.g., Walkeret al., Molec. Immunol. 26:403 (1989). The antibodies can be recombinantmonoclonal antibodies produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies canalso be chemically constructed according to the method disclosed in U.S.Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, Fab′, F(ab)₂, and Fv fragments; domainantibodies, diabodies; vaccibodies, linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. Such fragments can be produced by known techniques. Forexample, F(ab′)₂ fragments can be produced by pepsin digestion of theantibody molecule, and Fab fragments can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., Science 254:1275 (1989)). In some embodiments, the term“antibody fragment” as used herein may also include any proteinconstruct that is capable of binding a target pathogen or sperm andassociate with mucin to trap the target pathogen or sperm in mucus.

Antibodies of the invention may be altered or mutated for compatibilitywith species other than the species in which the antibody was produced.For example, antibodies may be humanized or camelized. Humanized formsof non-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions (i.e., the sequences between the CDR regions)are those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fe), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al.,Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593(1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain Humanization canessentially be performed following the method of Winter and co-workers(Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534 (1988)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues (e.g., all of theCDRs or a portion thereof) and possibly some FR residues are substitutedby residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be madeby introducing human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in thefollowing scientific publications: Marks et al., Bio/Technology 10:779(1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812(1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger,Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev.Immunol. 13:65 (1995).

Immunogens (antigens) are used to produce antibodies specificallyreactive with target polypeptides. Recombinant or synthetic polypeptidesand peptides, e.g., of at least 5 (e.g., at least 7 or 10) amino acidsin length, or greater, are the preferred immunogens for the productionof monoclonal or polyclonal antibodies. In one embodiment, animmunogenic polypeptide conjugate is also included as an immunogen. Thepeptides are used either in pure, partially pure or impure form.Suitable polypeptides and epitopes for target pathogens and sperm arewell known in the art. Polynucleotide and polypeptide sequences areavailable in public sequence databases such as GENBANK®/GENPEPT®. Largenumbers of neutralizing and non-neutralizing antibodies thatspecifically bind to target pathogens and sperm have been described inthe art and can be used as starting material to prepare the antibodiesof the present invention. Alternatively, new antibodies can be raisedagainst target pathogens and sperm using the techniques described hereinand well known in the art.

Recombinant polypeptides are expressed in eukaryotic or prokaryoticcells and purified using standard techniques. The polypeptide, or asynthetic version thereof, is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies can begenerated for subsequent use in immunoassays to measure the presence andquantity of the polypeptide.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, e.g., a purified or syntheticpeptide, a peptide coupled to an appropriate carrier (e.g.,glutathione-S-transferase, keyhole limpet hemanocyanin, etc.), or apeptide incorporated into an immunization vector such as a recombinantvaccinia virus is optionally mixed with an adjuvant and animals areimmunized with the mixture. The animal's immune response to theimmunogen preparation is monitored by taking test bleeds and determiningthe titer of reactivity to the peptide of interest. When appropriatelyhigh titers of antibody to the immunogen are obtained, blood iscollected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to thepeptide is performed where desired. Antibodies, including bindingfragments and single chain recombinant versions thereof, against thepolypeptides are raised by immunizing animals, e.g., using immunogenicconjugates comprising a polypeptide covalently attached (conjugated) toa carrier protein as described above. Typically, the immunogen ofinterest is a polypeptide of at least about 10 amino acids, in anotherembodiment the polypeptide is at least about 20 amino acids in length,and in another embodiment, the fragment is at least about 30 amino acidsin length. For example, the polypeptide can comprise amino acids acidresidues 1 through 200 from the N-terminal of the papillomavirus L2protein. The immunogenic conjugates are typically prepared by couplingthe polypeptide to a carrier protein (e.g., as a fusion protein) or,alternatively, they are recombinantly expressed in an immunizationvector.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies are screened for binding to normal ormodified peptides, or screened for agonistic or antagonistic activity.Specific monoclonal and polyclonal antibodies will usually bind with aK_(D) of at least about 50 mM, e.g., at least about 1 mM, e.g., at leastabout 0.1 mM or better. In some instances, it is desirable to preparemonoclonal antibodies from various mammalian hosts, such as mice,rodents, primates, humans, etc. Description of techniques for preparingsuch monoclonal antibodies are found in Kohler and Milstein 1975 Nature256:495-497. Summarized briefly, this method proceeds by injecting ananimal with an immunogen, e.g., an immunogenic peptide either alone oroptionally linked to a carrier protein. The animal is then sacrificedand cells taken from its spleen, which are fused with myeloma cells. Theresult is a hybrid cell or “hybridoma” that is capable of reproducing invitro. The population of hybridomas is then screened to isolateindividual clones, each of which secrete a single antibody species tothe immunogen. In this manner, the individual antibody species obtainedare the products of immortalized and cloned single B cells from theimmune animal generated in response to a specific site recognized on theimmunogenic substance.

Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe antigen, and yield of the monoclonal antibodies produced by suchcells is enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate (preferably mammalian) host. Thepolypeptides and antibodies of the present invention are used with orwithout modification, and include chimeric antibodies such as humanizedmurine antibodies. Other suitable techniques involve selection oflibraries of recombinant antibodies in phage or similar vectors. See,Huse et al. 1989 Science 246:1275-1281; and Ward et al. 1989 Nature341:544-546.

Antibodies specific to the target polypeptide can also be obtained byphage display techniques known in the art.

Antibodies can sometimes be labeled by joining, either covalently ornoncovalently, a substance which provides a detectable signal. A widevariety of labels and conjugation techniques are known and are reportedextensively in both the scientific and patent literature. Suitablelabels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Such antibodies are useful for detecting ordiagnosing the presence of a microbe on which an antigen is found.

Method of making antibodies with a glycosylation pattern of interest canbe achieved by any method known to those or skill in the art. Forexample, in some embodiments, mammalian cells can be used, such as,Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, andNS0- and SP2/0-mouse myeloma cells, to produce antibodies having thedesired glycosylation pattern. In certain embodiments, human cell linescan be used, e.g., 293 cells. In some embodiments, non-mammalian cellscan be used. The cell line can be genetically engineered to produce theantibodies with the desired oligosaccharide. Such cell lines can havealtered expression, for example, of one or more enzymes affectingglycosylation patterns, e.g., glycosyltransferases. Glycosyltransferasesinclude, without limitation, a galactosyltransferase, afucosyltransferase, a glucosyltransferase, anN-acetylgalactosaminyltransferase, an N-acetylglucosaminyltransferase, aglucuronyltransferase, a sialyltransferase, a mannosyltransferase, aglucuronic acid transferase, a galacturonic acid transferase, anoligosaccharyltransferase, or any combination thereof. Specific examplesinclude, without limitation, oligosaccharyltransferase,UDP-N-acetyl-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase, GDP-fucoseprotein:O-fucosyltransferase 1, GDP-fucose protein:O-fucosyltransferase2, protein:O-glucosyltransferase, UDP-N-acetylglucosamine:peptideN-acetylglucosaminyltransferase, protein:O-mannosyltransferase, β1,4galactosyltransferase, and any combination thereof. Enzymes involved inglycosylation of proteins are well known in the art and can bemanipulated using routine techniques. See, for example, U.S. Pat. Nos.8,383,106, 8,367,374, 8,080,415, 8,025,879, 8,021,856, 7,906,329, and7,846,434, each incorporated herein by reference in its entirety. Inother embodiments, glycans can be synthesized in specific patterns andlinked to antibodies. In further embodiments, antibodies with mixedglycosylation patterns can be separated to isolate antibodies with thedesired glycosylation pattern.

As would be recognized by one skilled in the art, the antibodies of thepresently-disclosed subject matter can also be formed into suitablecompositions, e.g., pharmaceutical compositions for administration to asubject in order to treat or prevent an infection caused by a targetpathogen or a disease or disorder caused by infection by a targetpathogen or to provide contraception. In one embodiment, thecompositions comprise, consist essentially of, or consist of an antibodyof the invention in a prophylactically or therapeutically effectiveamount and a pharmaceutically-acceptable carrier.

Pharmaceutical compositions containing the antibodies as disclosedherein can be formulated in combination with any suitable pharmaceuticalvehicle, excipient or carrier that would commonly be used in this art,including such conventional materials for this purpose, e.g., saline,dextrose, water, glycerol, ethanol and combinations thereof. As oneskilled in this art would recognize, the particular vehicle, excipientor carrier used will vary depending on the subject and the subject'scondition, and a variety of modes of administration would be suitablefor the compositions of the invention. Suitable methods ofadministration of any pharmaceutical composition disclosed in thisapplication include, but are not limited to, topical, oral, intranasal,buccal, inhalation, anal, and vaginal administration, wherein suchadministration achieves delivery of the antibody to a mucus membrane ofinterest.

The composition can be any type of composition suitable for deliveringantibody to a mucosal surface and can be in various forms known in theart, including solid, semisolid, or liquid form or in lotion form,either oil-in-water or water-in-oil emulsions, in aqueous gelcompositions. Compositions include, without limitation, gel, paste,suppository, douche, ovule, foam, film, spray, ointment, pessary,capsule, tablet, jelly, cream, milk, dispersion, liposomes, powder/talcor other solid, suspension, solution, emulsion, microemulsion,nanoemulsion, liquid, aerosol, microcapsules, time-release capsules,controlled release formulation, sustained release formulation orbioadhesive gel (e.g., a mucoadhesive thermogelling composition) or inother forms embedded in a matrix for the slow or controlled release ofthe antibody to the surface onto which it has been applied or incontact. Of particular interest herein are nebulized compositions,suitable for aerosol delivery.

The composition may contain conventional additives, such aspreservatives, solvents to promote penetration, and emollients. Topicalformulations may also contain conventional carriers such as cream orointment bases, ethanol, or oleyl alcohol. Other formulations foradministration, including intranasal administration, etc., arecontemplated for use in connection with the presently-disclosed subjectmatter. All formulations, devices, and methods known to one of skill inthe art which are appropriate for delivering the antibody or compositioncontaining the antibody to one or more mucus membranes of a subject canbe used in connection with the presently-disclosed subject matter.

The compositions used in the methods described herein may include otheragents that do not negatively impact or otherwise affect the inhibitoryand/or contraceptive effectiveness of the components of the composition,including antibodies, antimicrobial agents, and/or sperm-functioninhibitors. For example, solid, liquid or a mixture of solid and liquidpharmaceutically acceptable carriers, diluents, vehicles, or excipientsmay be employed in the pharmaceutical compositions. Suitablephysiologically acceptable, substantially inert carriers include water,a polyethylene glycol, mineral oil or petrolatum, propylene glycol,hydroxyethylcellulose, carboxymethyl cellulose, cellulosic derivatives,polycarboxylic acids, linked polyacrylic acids. such as carbopols; andother polymers such as poly(lysine), poly(glutamic acid), poly(maleicacid), polylactic acid), thermal polyaspartate, and aliphatic-aromaticresin; glycerin, starch, lactose, calcium sulphate dihydrate, terraalba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate,stearic acid, syrup, peanut oil, olive oil, saline solution, and thelike.

The pharmaceutical compositions described herein useful in the methodsof the present invention may further include diluents, fillers, bindingagents, colorants, stabilizers, perfumes, gelling agents, antioxidants,moisturizing agents, preservatives, acids, and other elements known tothose skilled in the art. For example, suitable preservatives are wellknown in the art, and include, for example, methyl paraben, propylparaben, butyl paraben, benzoic acid and benzyl alcohol.

Compositions can be powdered or liquid and may include one or moreinactive ingredients and/or carriers, such as glucose, lactose, sucrose,mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that can beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

The composition can comprise a powder or an aerosolized or atomizedsolution or suspension comprising the antibody. Such powdered,aerosolized, or atomized compositions, when dispersed, preferably havean average particle or droplet size in the range from about 0.1 to about200 nanometers.

The antibody can be formulated for nasal administration or otherwiseadministered to the lungs of a subject by any suitable means, e.g.,administered by an aerosol suspension of respirable particles comprisingthe antibody, which the subject inhales. The respirable particles can beliquid or solid. The term “aerosol” includes any gas-borne suspendedphase, which is capable of being inhaled into the bronchioles or nasalpassages. Specifically, aerosol includes a gas-borne suspension ofdroplets, as can be produced in a metered dose inhaler or nebulizer, orin a mist sprayer. Aerosol also includes a dry powder compositionsuspended in air or other carrier gas, which can be delivered byinsufflation from an inhaler device, for example. See Ganderton & Jones,Drug Delivery to the Respiratory Tract, Ellis Harwood (1987); Gonda(1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313;and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosolsof liquid particles comprising the antibody can be produced by anysuitable means, such as with a pressure-driven aerosol nebulizer,vibrating mesh nebulizer or an ultrasonic nebulizer, as is known tothose of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosolsof solid particles comprising the antibody can likewise be produced withany solid particulate medicament aerosol generator, by techniques knownin the pharmaceutical art.

Alternatively, one can administer the antibody in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

As noted herein, antibodies of the presently-disclosed subject matterare capable of diffusing through mucus when they are unbound, to allowthe antibody to bind a target pathogen or sperm at a desirable rate. Itis also desirable that, when antibodies are bound to the pathogen orsperm, the cumulative effect of the antibody-mucin interactionseffectively traps the pathogen or sperm in the mucus. To facilitate thisgoal, in some embodiments, it can be desirable to provide a compositionthat includes more than one antibody, wherein each antibody specificallybinds a different epitope of the target pathogen or sperm. Such acomposition provides the ability for an increased number of antibodiesto become bound to the pathogen or sperm, thereby strengthening theantibody-mucin interactions that serve to trap the pathogen or sperm inthe mucus.

In some embodiments of the presently-disclosed subject matter, acomposition includes a first antibody and a second antibody, asdisclosed herein, wherein the first antibody specifically binds a firstepitope of the target pathogen or sperm and the second antibodyspecifically binds a second epitope of the target pathogen or sperm,wherein said first epitope is distinct from the second epitope. Incertain embodiments, the composition includes three or more differentantibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies,wherein each antibody specifically binds a different epitope of thetarget pathogen or sperm.

In some embodiments of the presently-disclosed subject matter, acomposition includes a first antibody that specifically binds a gGsurface glycoprotein of HSV, a second antibody that specifically binds agD surface glycoprotein of HSV, and/or a third antibody thatspecifically binds a gB surface glycoprotein of HSV.

It is also desirable to provide a composition that can provide treatmentor prevention of infection due to more than one target pathogen. In someembodiments of the presently-disclosed subject matter, a compositionincludes a first antibody and a second antibody, as disclosed herein,wherein the first antibody specifically binds an epitope of a firsttarget pathogen and the second antibody specifically binds an epitope ofsecond target pathogen. In certain embodiments, the composition includesthree or more different antibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, ormore different antibodies, wherein each antibody specifically binds anepitope of a different target pathogen.

In other embodiments, a composition provides treatment or prevention ofinfection by one or more target pathogens. In some embodiments of thepresently-disclosed subject matter, a composition includes a firstantibody and a second antibody, as disclosed herein, wherein the firstantibody specifically binds an epitope of sperm and the second antibodyspecifically binds an epitope of a target pathogen. In certainembodiments, the composition includes three or more differentantibodies, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies,wherein one or more antibodies bind different epitopes of sperm and oneor more antibodies specifically binds an epitope of a target pathogen ormultiple target pathogens.

In some embodiments, the pharmaceutical composition can further includean additional active agent, e.g., a prophylactic or therapeutic agent.For example, the additional active agent can be an antimicrobial agent,as would be known to one of skill in the art. The antimicrobial agentmay be active against algae, bacteria, fungi, parasites (helminths,protozoa), viruses, and subviral agents. Accordingly, the antimicrobialagent may be an antibacterial, antifungal, antiviral, antiparasitic, orantiprotozoal agent. The antimicrobial agent is preferably activeagainst infectious diseases.

Suitable antiviral agents include, for example, virus-inactivatingagents such as nonionic, anionic and cationic surfactants, and C31 G(amine oxide and alkyl betaine), polybiguanides, docosanol,acylcarnitine analogs, octyl glycerol, and antimicrobial peptides suchas magainins, gramicidins, protegrins, and retrocyclins. Mildsurfactants, e.g., sorbitan monolaurate, may advantageously be used asantiviral agents in the compositions described herein. Other antiviralagents that may advantageously be utilized in the compositions describedherein include nucleotide or nucleoside analogs, such as tenofovir,acyclovir, amantadine, didanosine, foscarnet, ganciclovir, ribavirin,vidarabine, zalcitabine, and zidovudine. Further antiviral agents thatmay be used include non-nucleoside reverse transcriptase inhibitors,such as UC-781 (thiocarboxanilide), pyridinones, TIBO, nevaripine,delavirdine, calanolide A, capravirine and efavirenz. From these reversetranscriptase inhibitors, agents and their analogs that have shown poororal bioavailability are especially suitable for administration tomucosal tissue, in combination with antibodies and compositions of theinvention, to prevent sexual transmission of HIV. Other antiviral agentsthat may be used are those in the category of HIV entry blockers, suchas cyanovirin-N, cyclodextrins, carregeenans, sulfated or sulfonatedpolymers, mandelic acid condensation polymers, monoclonal antibodies,chemokine receptor antagonists such as TAK-779, SCH-C/D, and AMD-3100,and fusion inhibitors such as T-20 and 1249.

Suitable antibacterial agents include antibiotics, such asaminoglycosides, cephalosporins, including first, second and thirdgeneration cephalosporins; macrolides, including erythromycins,penicillins, including natural penicillins, penicillinase-resistantpenicillins, aminopenicillins, extended spectrum penicillins;sulfonamides, tetracyclines, fluoroquinolones, metronidazole and urinarytract antiseptics.

Suitable antifungal agents include amphotericin B, nystatin,griseofulvin, flucytosine, fluconazole, potassium iodide, intraconazole,clortrimazole, miconazole, ketoconazole, and tolnaftate.

Suitable antiprotozoal agents include antimalarial agents, such aschloroquine, primaquine, pyrimethamine, quinine, fansidar, andmefloquine; amebicides, such as dioloxamide, emetine, iodoquinol,metronidazole, paromomycine and quinacrine; pentamidine isethionate,atovaquone, and eflornithine.

The presently-disclosed subject matter further includes a kit, includingthe antibody or composition comprising the antibody as described herein;and optionally a device for administering the antibody or composition.In some embodiments, the kit can include multiple antibodies and/orcompositions containing such antibodies. In some embodiments, each ofmultiple antibodies provided in such a kit can specifically bind to adifferent epitope of the target pathogen or sperm. In other embodiments,each of multiple antibodies provided in such a kit can specifically bindto an epitope of a different target pathogen or to an epitope of sperm.In some embodiments, the kit can further include an additional activeagent, e.g., an antimicrobial, such as an antibiotic, an antiviral, orother antimicrobial, or a sperm-function inhibitor as would be known toone of skill in the art.

Prevention and Treatment of Infection

The presently-disclosed subject matter further includes methods ofinhibiting or treating an infection by a target pathogen in a subject,including administering to a mucosa of the subject an antibody and/orcomposition as disclosed herein. The mucosa can be selected from, forexample, a respiratory tract mucosa (e.g., a nasal mucosa, a lungmucosa), a reproductive tract mucosa (e.g., a genital mucosa, a uterinemucosa, a vaginal mucosa), an ocular mucosa, and a gastrointestinalmucosa (e.g., an oral mucosa, an anal mucosa), and any combinationthereof. In certain embodiments, the methods comprise additional stepssuch as one or more of isolating the antibodies, preparing a compositionof the isolated antibodies, determining the level of antibodies in themucus of the subject before administering the antibodies, anddetermining the level of antibodies in the mucus of the subject afteradministering the antibodies.

The antibodies and compositions of the present invention according tothe methods described herein are administered or otherwise applied bydelivering the composition, typically to a site of infection. The siteof infection may be one where an infection is already present (an actualsite of infection) or where an infection is likely to occur (a potentialsite of site of infection in or on an uninfected individual). In someembodiments, the antibodies and compositions may be topically delivered.In other embodiments, the antibodies and compositions may besystemically delivered such that the antibodies are secreted into themucus of the subject. Accordingly, the compositions as described abovemay be delivered the respiratory tract, e.g., the nasal cavity and thelungs.

An effective amount of the antibody can be administered. As used herein,an “effective amount” of the antibody for inhibition of infection refersto a dosage sufficient to inhibit infection by the target pathogen. Asused herein, an “effective amount” of the antibody for treatment ofinfection refers to a dosage sufficient to inhibit spread of the targetpathogen from infected cells to non-infected cells in the subject and/orto inhibit spread of the target pathogen from the infected subject toanother subject, e.g., a non-infected subject. The effective amount canbe an amount sufficient to trap an amount of the target pathogen inmucus. As will be recognized by one of skill in the art, the amount canvary depending on the patient and the target pathogen. The exact amountthat is required will vary from subject to subject, depending on thespecies, age, and general condition of the subject, the particularcarrier or adjuvant being used, mode of administration, and the like. Assuch, the effective amount will vary based on the particularcircumstances, and an appropriate effective amount can be determined ina particular case by one of skill in the art using only routineexperimentation. In some instances, an effective amount of the antibodythat specifically binds the target pathogen or sperm can be an amountthat achieves a concentration of the antibody in the mucus of about 0.1μg/mL to about 1000 μg/mL, e.g., about 0.5 μg/mL to about 100 μg/mL,e.g., about 1 μg/mL to about 50 μg/mL or any range therein. In someembodiments, the antibody may be administered in two or more stages withdifferent doses in each stage. For example, higher doses can beadministered initially in order to clear target pathogens that arepresent in the mucus of exposed or infected subjects and ensure thatsufficient amounts of antibody remain in the mucus to provideprotection, e.g., for about 24 hours. In later stages, lower doses canbe administered to maintain protective levels of the antibody. In otherembodiments, protective doses can be administered to subjects that arelikely to be exposed to a pathogen and higher doses can be administeredif infection occurs.

As will be recognized by one of skill in the art, the term “inhibiting”or “inhibition” does not refer to the ability to completely eliminatethe possibility of infection in all cases. Rather, the skilled artisanwill understand that the term “inhibiting” refers to reducing thechances of pathogens moving through mucus beyond the mucus membrane suchthat infection of a subject can occur, such as reducing chances ofinfection by a pathogen when such pathogen is bound to trappingantibodies in mucus. Such decrease in infection potential can bedetermined relative to a control that has not been administered theantibodies of the invention. In some embodiments, the decrease ofinhibition potential relative to a control can be about a 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% decrease.

In some embodiments inhibiting or treating an infection in a subject cancomprise trapping a pathogen in mucus. As such, in some embodiments amethod of trapping a target pathogen in mucus is provided, which methodincludes administering to a mucosa of the subject an antibody orcomposition as described herein.

In some embodiments, a method of inhibiting or treating an infection ina subject, and/or trapping a pathogen in the mucus of a subject,involves administering to a mucosa of the subject a compositioncomprising an isolated antibody that specifically binds anon-neutralizing epitope of a target pathogen. The antibody can be anon-neutralizing antibody. In some embodiments, the non-neutralizingantibody is provided at a concentration above a predetermined amount.

In some embodiments, a method of inhibiting or treating an infection ina subject, and/or trapping a pathogen in the mucus of a subject,involves administering to a mucosa of the subject a compositioncomprising an isolated antibody that specifically binds a neutralizingepitope of a target pathogen, wherein the antibody is provided at asub-neutralization dose.

As used herein, the term “subject” refers to humans and other animals.Thus, veterinary treatment is provided in accordance with thepresently-disclosed subject matter. As such, the presently-disclosedsubject matter provides for the treatment of mammals such as humans, aswell as those mammals of importance due to being endangered, such asSiberian tigers; of economic importance, such as animals raised onfarms; and/or animals of social importance to humans, such as animalskept as pets or in zoos. Examples of such animals include but are notlimited to: carnivores such as cats and dogs; swine, including pigs,hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen,sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, alsoprovided is the treatment of livestock, including, but not limited to,domesticated swine, ruminants, ungulates, horses (including racehorses), poultry, and the like.

A subject in need of inhibiting an infection or a disease or disordercaused by an infection is a subject that has been identified as being atrisk of infection. In some embodiments, the subject is identified ashaving been exposed to the target pathogen. In other embodiments, thesubject is in contact with other subjects that are infected or arelikely to become infected, e.g., a subject that is living with, workingwith, and/or attending school with infected subjects.

A subject in need of treating an infection or a disease or disordercaused by an infection is a subject that has been diagnosed as infectedwith a pathogen or is suspected of being infected with a pathogen, e.g.,exhibiting symptoms of infection.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) of the antibody,composition, or device comprising the composition can be employed over avariety of time intervals (e.g., hourly, daily, weekly, monthly, etc.)to achieve prophylactic and/or therapeutic effects.

In some embodiments, the method include administering an antibody, andfurther administering an additional active agent, e.g., prophylactic ortherapeutic agent, e.g., an antimicrobial, such as an antibiotic, anantiviral, or other antimicrobial as would be known to one of skill inthe art. The additional active agents can be delivered concurrently withthe antibodies and compositions of the invention. The additional activeagents can be delivered in the same composition as the antibody or inseparate compositions. As used herein, the word “concurrently” meanssufficiently close in time to produce a combined effect (that is,concurrently can be simultaneously, or it can be two or more eventsoccurring within a short time period before or after each other).

Monitoring Vaccine Effectiveness

Having discovered the usefulness of the antibodies of the invention inpreventing and treating infection by pathogens and/or diseases ordisorders caused by infection by pathogens, another aspect of theinvention relates to detecting the presence of such antibodies in asubject in order to determine how well the subject is protected againstpossible infection. For example, a subject that has been vaccinatedagainst a particular pathogen can be monitored for the presence andlevel of antibodies that have trapping potency in mucus or other bodyfluids, e.g., saliva. If such antibodies do not appear in the mucus orappear at non-prophylactic or non-therapeutic levels, the subject can beadministered a booster vaccination and/or a different vaccine in orderto generate an effective amount of antibodies. For example, subjects canbe monitored to ensure a level of antibodies in mucus that at leastequal to the level of antibodies observed in a subject that has beenexposed previously to a pathogen.

Thus, one embodiment relates to a method of monitoring the effectivenessof a vaccination in a subject that has been vaccinated against a targetpathogen, comprising determining in a mucus sample from said subject theamount of an antibody comprising an oligosaccharide at a glycosylationsite, the oligosaccharide having a glycosylation pattern that enhancestrapping potency of the antibody in mucus, wherein the antibodyspecifically binds an epitope of said target pathogen, wherein theamount of said antibody is indicative of the effectiveness of thevaccination.

The mucus to be sampled can be from the appropriate mucosal surfacewhere the antibody is expected to be found. Samples of mucus can beobtained from a subject by methods routinely used in the art. Similarly,samples of other body fluids such as saliva can be obtained from asubject by methods routinely used in the art.

A variety of assays can be employed for detection and/or quantitation ofthe antibody. For example, various immunoassays can be used to detectantibodies of this invention. Such immunoassays typically involve themeasurement of antigen/antibody complex formation between a protein orpeptide (i.e., an antigen) and its specific antibody.

The immunoassays of the invention can be either competitive ornoncompetitive and both types of assays are well-known andwell-developed in the art. In competitive binding assays, antigen orantibody competes with a detectably labeled antigen or antibody forspecific binding to a capture site bound to a solid surface. Theconcentration of labeled antigen or antibody bound to the capture agentis inversely proportional to the amount of free antigen or antibodypresent in the sample.

Noncompetitive assays of this invention can be, for example, sandwichassays, in which, for example, the antigen is bound between twoantibodies. One of the antibodies is used as a capture agent and isbound to a solid surface. The other antibody is labeled and is used tomeasure or detect the resultant antigen/antibody complex by e.g., visualor instrument means. A number of combinations of antibody and labeledantibody can be used, as are well known in the art. In some embodiments,the antigen/antibody complex can be detected by other proteins capableof specifically binding human immunoglobulin constant regions, such asprotein A, protein L or protein G. These proteins are normalconstituents of the cell walls of streptococcal bacteria. They exhibit astrong nonimmunogenic reactivity with immunoglobulin constant regionsfrom a variety of species. (See, e.g., Kronval et al., J. Immunol.,111:1401 (1973); Akerstrom et al., J. Immunol., 135:2589 (1985)).

In some embodiments, the non-competitive assays need not be sandwichassays. For instance, the antibodies or antigens in the sample can bebound directly to the solid surface. The presence of antibodies orantigens in the sample can then be detected using labeled antigen orantibody, respectively.

In some embodiments, antibodies and/or proteins can be conjugated orotherwise linked or connected (e.g., covalently or noncovalently) to asolid support (e.g., bead, plate, slide, dish, membrane or well) inaccordance with known techniques. Antibodies can also be conjugated orotherwise linked or connected to detectable groups such as radiolabels(e.g., ³⁵S, ¹²⁵I, ³²P, ¹³H, ¹⁴C, ¹³¹D, enzyme labels (e.g., horseradishperoxidase, alkaline phosphatase), gold beads, chemiluminescence labels,ligands (e.g., biotin) and/or fluorescence labels (e.g., fluorescein) inaccordance with known techniques.

A variety of organic and inorganic polymers, both natural and syntheticcan be used as the material for the solid surface. Nonlimiting examplesof polymers include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon,nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF),silicones, polyformaldehyde, cellulose, cellulose acetate,nitrocellulose, and the like. Other materials that can be used include,but are not limited to, include paper, glass, ceramic, metal,metalloids, semiconductive materials, cements and the like. In addition,substances that form gels, such as proteins (e.g., gelatins),lipopolysaccharides, silicates, agarose and polyacrylamides can be used.Polymers that form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids, long chain(12-24 carbon atoms) alkyl ammonium salts and the like are alsosuitable. Where the solid surface is porous, various pore sizes can beemployed depending upon the nature of the system.

A variety of immunoassay systems can be used, including but not limitedto, radio-immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA)assays, enzyme immunoassays (EIA), “sandwich” assays, gel diffusionprecipitation reactions, immunodiffusion assays, agglutination assays,immunofluorescence assays, fluorescence activated cell sorting (FACS)assays, immunohistochemical assays, protein A immunoassays, protein Gimmunoassays, protein L immunoassays, biotin/avidin assays,biotin/streptavidin assays, immunoelectrophoresis assays,precipitation/flocculation reactions, immunoblots (Western blot;dot/slot blot); immunodiffusion assays; liposome immunoassay,chemiluminescence assays, library screens, expression arrays,immunoprecipitation, competitive binding assays and immunohistochemicalstaining. These and other assays are described, among other places, inHampton et al. (Serological Methods, a Laboratory Manual, APS Press, StPaul, Mimi (1990)) and Maddox et al. (J. Exp. Med. 158:1211 (1993); theentire contents of which are incorporated herein by reference forteachings directed to immunoassays).

The methods of this invention can also be carried out using a variety ofsolid phase systems, such as described in U.S. Pat. No. 5,879,881, aswell as in a dry strip lateral flow system (e.g., a “dipstick” system),such as described, for example, in U.S. Patent Publication No.2003/0073147, the entire contents of each of which are incorporated byreference herein.

The detection and/or quantitation of the antibody in the mucus samplecan further comprise characterization of the glycosylation pattern ofthe antibody. Determination of the glycosylation pattern can be carriedout using methods well known in the art and described herein, includingglycan sequencing and separation techniques such as gel electrophoresis,fluorescence-activated sorting, and mass spectrometry.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Materials and Methods

Culture and Purification of Fluorescent HSV-1: The HSV-1 mutant 166v(Elliott et al., J. Virol. 73:4110 (1999)), encoding a VP22-GreenFluorescent Protein (GFP) tegument protein packaged into HSV-1 atrelatively high copy numbers (Heine et al., J Virol. 14:640 (1974)), waskindly provided by Richard Courtney and used in all microscopy and ELISAstudies, besides those for in vivo experiments. The addition of GFP tothe VP22 protein appears to have no deleterious effects on viralreplication (Elliott et al., J. Virol. 73:4110 (1999)), and thefluorescence of 166v was consistently more intense than that of HSV-1mutants encoding other GFP fusion proteins. 166v was expanded at an MOIof 3 on confluent monolayers of HaCat cells maintained in DMEM (LifeTechnologies, Grand Island, N.Y.) supplemented with 5% FBS,1×L-glutamine and 1× Penicillin/Streptomycin. Culture medium wascollected 16-18 hr post-infection and twice centrifuged at 1000×g for 5min to remove cell debris. The resulting supernatant was split into 30mL aliquots and precipitated overnight with a polyethylene glycol(PEG)/salt solution. Briefly, 10 mL of 1.55 M NaCl was added to 30 mL ofcrude virus supernatant, followed by 10 mL of 40% PEG 8000 (Sigma, St.Louis, Mo.). After an overnight incubation at 4° C. the virus/PEGsolution was centrifuged at 2555×g and 4° C. for 1 hr. The virus pelletwas then resuspended in 1×PBS and centrifuged through a continuous20-50% (w/w) sucrose in PBS gradient for 1 hr at 74,119×g. The resultingvirus band was collected, diluted 1:5 in PBS, layered over 30% (w/w)sucrose in PBS, and centrifuged for 1.5 hr at 83,472×g to pellet virusfor further purification. Purified virus pellet was resuspended in PBSand stored as single use aliquots at −80° C.

Cervicovaginal Mucus (CVM) Collection and Characterization: CVMcollection was performed as published previously (Lai et al., J. Virol.83:11196 (2009); Lai et al., Proc. Natl. Acad. Sci. U.S.A. 107:598(2010)). Briefly, undiluted CVM secretions, averaging 0.3 g per sample,were obtained from women of reproductive age, ranging from 20 to 32years old (27.4±0.9 years, mean±SEM), by using a self-sampling menstrualcollection device following protocols approved by the InstitutionalReview Board of the University of North Carolina—Chapel Hill. Informedconsent of participants was obtained after the nature and possibleconsequences of the study were explained. Participants inserted thedevice into the vagina for at least 30 s, removed it, and placed it intoa 50 mL centrifuge tube. Samples were centrifuged at 230×g for 2 min tocollect the secretions. Aliquots of CVM for lactic acid and Abmeasurements (diluted 1:5 with 1×PBS and stored at −80° C.) and slidesfor gram staining were prepared immediately, and the remainder of thesample was stored at 4° C. until microscopy, typically within a fewhours. Samples were collected at random times throughout the menstrualcycle, and cycle phase was estimated based on the last menstrual perioddate normalized to a 28 day cycle. No samples were ovulatory based onvisual observation (none exhibited spinnbarkeit). Samples that werenon-uniform in color or consistency were discarded. Donors stated theyhad not used vaginal products nor participated in unprotectedintercourse within 3 days prior to donating. All samples had pH<4.5;none had bacterial vaginosis (BY) based on Gram staining and Nugentscoring, following scoring criteria described previously (Nugent et al.,J. Clin. Microbiol. 29:297 (1991)). For lactic acid and Ab measurements,CVM aliquots were thawed and centrifuged for 2 min at 21,130×g to obtaincell-free supernatant containing lactic acid and Ab. Lactic acid contentwas measured using a DA-lactic acid kit (R-Biopharm, Darmstadt, Germany)according to manufacturer protocol, but adapted to a 96-well format.

Total immunoglobulin levels in CVM were quantified using the HumanIsotyping Kit (HGAMMAG-301K; Millipore, Billerica, Mass.) according tomanufacturer protocol. Briefly, 20× stock isotyping beads were vortexed,sonicated, diluted to IX, and incubated with 50 μL of serially dilutedCVM supernatant at 1:2 beads:CVM volume ratio. After 1 hr, the beadswere separated from CVM supernatant using a magnetic plate, and washedtwice with wash buffer. The beads were then incubated with 25 μL of 1×anti-Human Kappa and Lambda-PE for 1 hr, washed twice, and resuspendedin Luminex Drive fluid. Fluorescence intensities indicative ofimmunoglobulin levels present in CVM were measured using the LuminexMAGPIX system, and data analysis was performed using Milliplex Analyst(v3.5.5.0; Vigene Tech Inc., Carlisle, Mass.). All incubations werecarried out at room temperature in the dark with vigorous agitation.

Whole-virus ELISA was used to quantify HSV-1 specific IgG. Briefly,high-affinity 96-well half-area plates (Thermo Scientific, Rockford,Ill.) were coated overnight at 4° C. with 25 μL per well ofaffinity-purified intact HSV-1 at 20 μg/mL (measured using BCA assay).The plates were washed four times with 0.05% Tween in PBS (PBS-T),blocked with 5% milk for at least 1 hr followed by PBS-T washes, thenincubated for at least 1 hr with serial dilutions of CVM supernatant.Following PBS-T washes, virion-bound IgG was quantified using F(ab′)₂anti-human IgG Fc (Goat)-HRP conjugate (709-1317; Rockland,Gilbertsville, Pa.) and 1-Step Ultra TMB substrate (Thermo Scientific,Rockford, Ill.), and compared to a standard generated on the same plateusing twice-purified anti-HSV-1 IgG, which was assumed to be >90% pure.TMB conversion was terminated with 2 N sulfuric acid, and absorbance wasmeasured at 450 nm using a BioTek Synergy 2 plate reader. HSV-1 specificIgA and IgM levels were too low to be detected by this assay.

Preparation and Characterization of Anti-HSV-1 IgG: Anti-HSV-1 IgG waspurified from intravenous immunoglobulin (IVIg, Privigen®; ≥98% IgG; CSLBehring, King of Prussia, Pa.) by affinity column purification. BrieflyHSV glycoproteins were extracted from purified HSV-1 by overnightincubation with Triton X-100 (final concentration 0.05%) at 4° C.,followed by centrifugation at 21,130×g and 4° C. for 1.5 hr. Theresulting supernatant containing HSV glycoproteins was coupled toAminoLink Plus Coupling Resins (Thermo Scientific, Rockford, Ill.)according to manufacturer protocol, and stored at 4° C. until use. Toextract HSV-1 specific IgG, the column was first warmed to roomtemperature and washed twice with 3 mL of equilibrium buffer (150 mMNaCl, 0.05% Tween, 10 mM sodium phosphate at pH 6): The column was thenloaded with 2 mL of IVIg buffer exchanged into equilibrium buffer usinga 50 kDa MWCO concentrator (Corning, Tewksbury, Mass.), and incubated atroom temperature for at least 45 min with end-over-end mixing. Unbound,non-specific Ab was removed by washing with 1 mL of equilibrium bufferfollowed by three additional rounds of wash buffer (4 mL each round; 500mM NaCl, 0.05% Tween, 10 mM sodium phosphate at pH 6). Bound Ab was theneluted using IgG Elution Buffer (Thermo Scientific, Rockford, Ill.);each elution consisted of three 3 mL volumes of elution buffer, and wascollected into tubes containing 100 μL of 10 mM sodium phosphate pH 6 toneutralize the elutions. Elutions from multiple runs were pooledtogether, concentrated and buffer exchanged into PBS using a 30 kDa MWCOconcentrator tube, supplemented with sodium azide (final concentration0.03%) and stored at 4° C. until use. A single purification roundremoved over 99% of non-specific IgG, tested by spiking IVIg with mouseIgG. Final concentration of purified IgG was measured via sandwichELISA, with plates coated with 2 μg/mL of anti-human IgG Fc (AlphaDiagnostics, San Antonio, Tex.) followed by detection with F(ab′)₂anti-human IgG Fc (Goat)-HRP, and compared to a standard curve generatedwith serial dilutions of stock IVIg.

Preparation and Characterization of Anti-HSV-1 F(Ab′)₂: HSV-1 specificIgG was fragmented using a F(ab′)₂ Preparation Kit (Thermo Scientific,Rockford, Ill.). Briefly, IgG was desalted into IgG Digestion Bufferusing 3 kDa MWCO concentrator tubes (Amicon 0.5 mL; Millipore,Billerica, Mass.) and incubated with pepsin at 37° C. for 3.5 hr withend-over-end mixing, followed by purification using a Protein A column(0.2 mL resin; Thermo Scientific, Rockford, Ill.) and PBS washes toremove undigested IgG. Fragmentation was confirmed using a non-reducing4-12% Bis-Tris gel with MOPS running buffer. The gel was stained withCoomassie Blue for imaging; note that the gel image presented in FIG. 3was desaturated and contrast-adjusted using Pixlr photo editor toproduce a black and white image (same settings applied across entireimage). A competitive inhibition ELISA was also performed to confirmthat the purified F(ab′)₂ blocked binding of intact anti-HSV-1 IgG.HSV-1 coated wells were incubated with either 1% milk or differentdilutions of anti-HSV-1 F(ab′)₂ for 1 hr, followed by incubation withintact HSV-1 specific IgG for 1 hr and quantification of bound IgG usingF(ab)₂ anti-human IgG Fc (Goat)-HRP conjugate. Total amounts ofanti-HSV-1 F(ab′)₂ were quantified via sandwich ELISA with plates coatedwith HSV-1 virions as described above, but with detection by anti-humanIgG F(ab′)₂ (Goat)-HRP conjugate (Rockland 209-1304).

Preparation and Characterization of Deglycosylated Anti-HSV-1 IgG:N-linked oligosaccharides on purified anti-HSV-1 IgG were cleaved byincubating 200 μg of IgG with 10 μL of PNGase and 11 tit of 10×G7reaction buffer (New England Biolabs, Ipswich, Mass.) for at least 24 hrat 37° C. IgG was then recovered using a Protein A column, eluted withIgG Elution Buffer into 100 μL of 10 mM sodium phosphate pH 6 and bufferexchanged into PBS using a 30 kDa MWCO concentrator tube. Thedeglycosylated IgG was further purified using a 0.8 mL spin columnimmobilized with Con A-agarose slurry (Vector Labs, Burlingame, Calif.)and washed thrice with equilibrium buffer (10 mM HEPES with 0.15 M NaClat pH 7.5) prior to incubation with end-over-end mixing for 45 min atroom temperature. Following the incubation, the column was spun at5000×g for 1 min to collect the flow through, which contains thedeglycosylated IgG, and washed thrice with equilibrium buffer tomaximize recovery. The flow through and the washes were pooled, bufferexchanged into PBS using a 30 kDa MWCO concentrator tube, supplementedwith sodium azide (final concentration 0.03%) and stored at 4° C. untiluse. Total amounts of deglycosylated IgG were measured as describedabove. Deglycosylation was confirmed using a lectin-ELISA assay.Briefly, high-affinity 96-well plates were coated overnight at 4° C.with 50 μL per well of 1 μg/mL purified deglycosylated HSV-1 IgG. Theplates were washed 4× with PBS-T, blocked with 300 μL/well of 1× CarboFree solution (Vector Labs, Burlingame, Calif.) for at least 1 hrfollowed by PBS-T washes, then incubated with 50 μL/well of 1 μg/mLbiotinylated Con A lectin (Vector Labs, Burlingame, Calif.) for at least2 hr. Following PBS-T washes, IgG-bound lectin was quantified usinganti-biotin-HRP conjugate (Vector Labs, Burlingame, Calif.) and 1-StepUltra TMB substrate, and compared to wells coated with affinity-purifiedHSV-1 IgG or IVIg. TMB conversion was terminated with 2 N sulfuric acid,and absorbance was measured at 460 nm using a BioTek Synergy 2platereader and normalized to the amount of IgG bound to coated wellsquantified using F(ab′)₂ anti-human IgG Fc (Goat)-HRP conjugate.

Neutralization Assay: Purified HSV-1 (^(˜)550 PFU; 5 μL) was incubatedwith 95 μL of HSV-1 specific IgG solution at different finalconcentrations for 1 hr with end-over-end mixing. The mixture was thendiluted with 210 μL of media, of which duplicate 150 μL aliquots weretransferred to confluent Vero cell monolayers in a 6-well plate. Plateswere incubated at 37° C. for 1 hr with periodic rocking to ensure thatthe plates did not dry out, before the HSV-1/Ab mixture was aspiratedoff and wells were washed with 2 mL of PBS. The plates were thenincubated for 3 days at 37° C. in 2% carboxymethyl cellulose in EMEMsupplemented with 1×L-glutamine and 1× Penicillin/Streptomycin, beforestaining with 1% crystal violet solution, and the resulting plaques weremanually counted and compared to control wells to determine the extentof neutralization.

Multiple Particle Tracking of HSV-1 in CVM: To mimic neutralization ofCVM by alkaline seminal fluid, we titrated CVM to pH 6.8-7.1 using smallvolumes C3% v/v) of 3 N NaOH, and confirmed pH using a micro pHelectrode (Microelectrodes, Inc., Bedford, N.H.) calibrated to pH 4, 7and 10 buffers. Samples were either untreated or treated by addition ofknown amounts of purified anti-HSV-1 IgG or control (anti-biotin) IgG.Control beads consisted of red or green fluorescent 200 nmcarboxyl-modified polystyrene particles (Molecular Probes, Eugene,Oreg.), either uncoated (PS; muco-adhesive) or covalently conjugatedwith low molecular weight (2 kDa), amine-functionalized polyethyleneglycol (PEG; Rapp Polymere, Tuebingen, Germany) to produce coatedparticles (PS-PEG; muco-inert), as previously described (Lai et al.,Proc. Natl. Acad. Sci. U.S.A. 104:1482 (2007)). Fluorescent virions orcontrol beads (approximately 10⁸-10⁹ particles/mL) were added at 5% v/vto 20 μL of CVM placed in a custom-made glass chamber, and incubated for1 hr at 37° C. prior to microscopy. The translational motions of theparticles were recorded using an EMCCD camera (Evolve 512; Photometrics,Tucson, Ariz.) mounted on an inverted epifluorescence microscope(AxioObserver D1; Zeiss, Thornwood, N.Y.), equipped with an AlphaPlan-Apo 100×/1.46 NA objective, environmental (temperature and CO₂)control chamber and an LED light source (Lumencor Light EngineDAPI/GFP/543/623/690). Videos (512×512, 16-bit image depth) werecaptured with MetaMorph imaging software (Molecular Devices, Sunnyvale,Calif.) at a temporal resolution of 66.7 ms and spatial resolution of 10nm (nominal pixel resolution 0.156 μm/pixel) for 20 s. The trackingresolution was determined by tracking the displacements of particlesimmobilized with a strong adhesive, following a previously describedmethod (Apgar et al., Biophys. J. 79:1095 (2000)). Particle trajectorieswere analyzed using MetaMorph software as described previously (Lai etal., Proc. Natl. Acad. Sci. U.S.A. 104:1482 (2007); Lai et al., J Virol.83:11196 (2009); Lai et al., Proc. Natl. Acad. Sci. U.S.A. 107:598(2010)); image contrast was adjusted to improve particle visibility, butthe same contrast level was applied throughout the entire video and didnot bias toward any particle population. Sub-pixel tracking resolutionis obtained by determining the precise location of the particle centroidby light-intensity-weighted averaging of neighboring pixels. Trappedparticles were defined as those with effective diffusivity(D_(eff))<0.01 μm²/s at a time scale (t) of 1 s (i.e., particles moveless than their diameter within 1 s). In a subset of experiments, it wasconfirmed that particles defined as trapped over the course of 20 sbased on this criterion remain confined to the same locations over morethan 15 min. The slope, α, of the log-log mean square displacement(<MSD>) vs. time scale plot provides a further measure of particlemobility: α=1 for pure unobstructed Brownian diffusion, e.g., particlesin water, a becomes smaller as obstruction to particle diffusionincreases, and a is zero for permanently trapped particles. At leastfive independent experiments in CVM from different donors, with n≥100particles per experiment, were performed for each condition. For asubset of donors, similar observations were made at least twice insamples obtained on separate days to ensure reproducibility, but onlyone sample was used for analysis.

Mouse Vaginal HSV-2 Challenge Model: All experiments conducted with micewere performed in accordance with protocols approved by the JohnsHopkins University Animal Care and Use Committee satisfying therequirements of the E.E.C. Guidelines (1986) and U.S. Federal Guidelines(1985). Female CF-1 mice (6-8 weeks old; Harlan, Frederick, Md.) weretreated with Depo-Provera′ (medroxyprogesterone acetate, 2.5 mg/mouse)by subcutaneous injection into the right flank 6-8 days prior to use,Depo-Provera® synchronizes mice in a prolonged diestrus-like state, inwhich the vaginal epithelium thins and susceptibility of the tissue toinfection increases (Cone et al., BMC Infect. Dis. 6:90 (2006)).Depo-Provera®-treated mice were randomly divided into groups of ten. Themouse vagina is pH neutral (Meysick et al., J. Parasitol. 78:157(1992)); therefore, no attempt was made to modify vaginal pH prior toinoculation. Inocula were prepared by mixing HSV-2 (final dose 2 ID₅₀;strain G, ATCC, Manassas, Va.) with medium or different concentrationsof control (anti-biotin) or anti-gG IgG (8.F.141; Santa CruzBiotechnology, Inc., Santa Cruz, Calif.), and incubating for 1 hr at 37°C. Mice were inoculated with 20 μL, of HSV-2 solution, delivered to thevagina using a 50 μL Wiretrol (Drummond, Broomall, Pa.), fire-polishedto avoid damage to the vaginal epithelium. In some studies, the mousevagina was gently washed with ^(˜)10 mL of normal saline delivered at 1mL/min through a smooth ball-tipped gavage needle connected to a syringepump, prior to HSV-2 challenge. Removal of mucus by this process wasmeasured using a fluorimetrie mucin assay, as previously described(Crowther et al., Anal. Biochem. 163:170 (1987)). Importantly, thegentle wash did not damage the vaginal epithelium, as confirmed bymicroscopy with a fluorescence-based dead cell stain (YOYO-1) thatassesses membrane integrity (Cone et al., 2006), compared toconventional lavage and/or vaginal swabbing with a cotton tip, whichinduced significant epithelial damage. YOYO-1 has been previously usedto reveal toxicity caused by detergent-based microbicides that led toincreased susceptibility to HSV infection (Cone et al., BMC Infect. Dis.6:90 (2006)). Tissue sectioning and H&E staining was performed by theAnimal Histopathology lab at the University of North Carolina—ChapelHill. Infection was assayed three days post-inoculation by detection ofvirus in vaginal lavages. Briefly, 50 μL of medium was pipetted in andout of the vagina 20 times, diluted to 0.2 mL and placed on target cells(ELVIS® HSV Test System; Diagnostic Hybrids, Athens, Ohio); infectedcells (foci) were identified one day later, following manufacturerprotocol. Scores for virus shedding were assigned on a scale of 0-4based on the approximate density of foci observed (“0”: 0; “0.5”: <100;“1”: 100-500; “2”: 500-1000; “3”: 1000+; “4”; saturated). At least threeindependent experiments were performed for each condition, with n=10animals per experimental group (n≥30 total).

Statistical Analysis: correlation between endogenous anti-HSV-1 IgGlevels and average particle or virus D_(eff) in individual CVM sampleswas measured using Pearson's correlation coefficient (r). Statisticalcomparisons were limited to two groups (test group compared with theappropriate control group performed during the same experiment).Fisher's exact test was used to determine the statistical significanceof reductions in % mice infected. A two-tailed Student's t-test (pairedfor comparisons of Ab-treated vs, native CVM for the same CVM samples)was used for all other comparisons. Differences were deemed significantat an alpha level of 0.05. All values are reported as mean±SEM unlessotherwise indicated.

Example 2 Trapping of HSV in Cervicovaginal Mucus

The hypothesis of trapping-in-mucus was explored using HSV-1 (d^(˜)180nm), a highly prevalent sexually transmitted virus. Fresh, undiluted CVMwas obtained predominantly from donors with normallactobacillus-dominated vaginal microbiota, as confirmed by Nugentscoring (Table 1). HSV-1 virions expressing a VP22-GFP tegument proteinconstruct, packaged at high copy numbers while maintaining native viralenvelope integrity, were mixed into CVM pH-neutralized to mimicneutralization by alkaline seminal fluid. Time-lapse microscopy ofvirion motions in real-time was then performed with high spatiotemporalresolution, and virion mobility was quantified using multiple particletracking over a long time scale. Substantial differences in HSV-1mobility were observed in CVM samples from different donors (FIG. 1A) in7 of 12 CVM samples, most virions diffused distances spanning severalmicrons over the course of 20 s, whereas in the remaining 5 CVM samples,the majority′ f virions were essentially trapped, moving less than theirdiameter (<200 nm) in 20 s.

TABLE 1 Characterization of CVM samples: menstrual cycle phase, Nugentscore and % lactic acid. Donor Cycle Cycle Nugent % ID day¹ phase³score⁴ Lactic acid⁵ F10 17 Luteal 1 2.4 ± 0.039 F12 25 Luteal 2 0.81 ±0.081  F14 26 Luteal 2 0.95 ± 0.10  F18 11 Follicular 4 0.56 ± 0.022  F9N/A² N/A 0 0.93 ± 0.062  F13  9 Follicular 0 1.1 ± 0.031 F15 25 Luteal 01.1 ± 0.042 F17 N/A N/A 1 1.2 ± 0.088 F2 15 Luteal 2 0.74 ± 0.073  F21N/A N/A 0 1.5 ± 0.081 F5 10 Follicular 0 1.4 ± 0.074 F8 19 Luteal 0 1.9± 0.15  Median 0.5 1.10 SEM 0.37 0.15 Donor Cycle Cycle Nugent % ID day¹phase³ score⁴ Lactic acid⁵ F10 17 Luteal 1 2.4 ± 0.039 F12 25 Luteal 22.81 ± 0.081  F14 26 Luteal 2 0.95 ± 0.10  F18 11 Follicular 4 0.56 ±0.022  F9 N/A² N/A 0 0.93 ± 0.062  F13  9 Follicular 0 1.1 ± 0.031 F1525 Luteal 0 1.1 ± 0.031 F17 N/A N/A 1 1.2 ± 0.088 F2 15 Luteal 2 0.74 ±0.073  F21 N/A N/A 0 1.5 ± 0.081 F5 10 Follicular 0 1.4 ± 0.074 F8 19Luteal 0 1.9 ± 0.15  Median 0.5 1.10 SEM 0.37 0.15 ¹Cycle day calculatedas the number of days from the first day of the last menstrual periodnormalized by the cycle length to a 28 day cycle. ²N/A= hormonalcontraceptive. ³Cycle phase estimated based on normalized cycle day; nosamples were ovulatory based on absence of spinnbarkeitby visualinspection. ⁴A Nugent score of 0-3 corresponds to “normal”(lactobacilli-dominated) microflora, 4-6 to “intermediate”, and 7-10 to“bacterial vaginosis” (BV)-a condition associated with greater risk ofSTI acquisition. Assessment of Nugent scores was independently confirmedby the Clinical Microbiology and Immunology Lab at UNC. ⁵Values areexpressed as a mean ± SEM. Grey highlight indicates CVM samplescontaining sufficient native levels of anti-HSV-1 IgG to immobilizevirions.

Since IgG is the predominant immunoglobulin in human CVM (Usala et al.,J. Reprod. Med. 34:292 (1989)), it was examined whether virion mobilitycorrelated with endogenous virus-specific IgG in all 12 CVM samplesusing a whole-virus ELISA assay (Table 2). In good agreement with thehypothesis, HSV-1 diffused rapidly through all CVM samples that hadlittle or no detectable endogenous anti-HSV-1 IgG (<0.2 μg/mL; detectionlimit 0.017 μg/mL) at rates only several-fold lower than their expectedrates in water (FIGS. 1 and 2A). In contrast, in samples with elevatedlevels of endogenous anti-HSV-1 IgG (≥0.6 μg/mL), most HSV-1 virionswere effectively trapped. HSV-1 that was trapped in place over the first20 s observation remained trapped in the same locations for at least 15min (FIG. 2B). In the same CVM samples, control latex nanoparticlescomparable in size to HSV-1 and engineered with muco-inert coatings(PS-PEG; d˜200 nm) exhibited rapid diffusion (FIGS. 1 and 2A), in goodagreement with previous observations of the large pores present in humanCVM (average ˜340 nm) ((Lai et al., Proc. Natl. Acad. Sci. U.S.A.104:1482 (2007); Lai et al., Proc. Natl. Acad. Sci. U.S.A. 107:598(2010)). Thus, the mucus mesh spacing was large enough for IgG-coatedHSV-1 (at most 15-20 nm larger diameter even at saturation) to diffuserelatively unimpeded in the absence of adhesive interactions with mucingel. Muco-adhesive latex nanoparticles of the same size (PS; d^(˜)200nm) were markedly slowed or immobilized in the same CVM secretions(FIGS. 1 and 2A). Importantly, observations with PS-PEG and PS controlparticles confirmed that the general barrier properties of all samples,including those with low levels of endogenous anti-HSV-1 IgG, remainedintact. HSV-1 mobility correlated only with endogenous HSV-1 specificIgG, and did not correlate with total IgG, IgA or IgM content (FIG. 3),After removal of ^(˜)90-95% of total IgG from these samples by dialysisat constant sample volume, HSV-1 became readily mobile (FIG. 4A),whereas PS beads remained immobilized (FIG. 4A).

TABLE 2 Characterization of CVM samples: Ab content. Anti-HSV-1 IgGDonor % of Total Total Total ID Average total IgG₁ IgG₂ IgG₃ IgG₄ IgGIgA IgM F10 0.92 ± 0.20  0.60% 90% 6.7%  3.4% N.D.² 150 ± 12 1.0 ± 0.222.1 ± 1.7 F12 0.66 ± 0.037 0.19% 65% 28% 8.1% 0.66% 360 ± 36 8.1 ± 1.1  1.2 ± 0.91 F14 1.9 ± 0.20 0.14% 78% 19% 4.2% 1.0% 1300 ± 130 79 ± 9.3  4.8 ± 0.11 F18 8.5 ± 0.31 0.56% 71% 20% 6.1% 4.5% 1500 ± 110 200 ± 15  72 ± 6.9 F9 23 ± 1.4  1.2% 83% 11% 4.0% 1.5% 1900 ± 250 150 ± 21   39 ±5.5 Median 1.9  0.56% 78% 19% 4.2% 1.0% 1300  79   4.8 SEM 4.3  0.19%4.3%  3.7%  0.88%  0.77% 340 38   14   F13  0.026 ± 0.00049 0.027% 76%18% 5.7% 0.28% 980 ± 79 7.0 ± 0.45 2.9 ± 1.3 F15 0.080 ± 0.0011 0.024%68% 29% 4.1% 0.28% 330 ± 43 5.0 ± 0.91  1.4 ± 0.92 F17 0.19 ± 0.0230.036% 49% 34%  20% 0.67% 540 ± 33 17± 1.5  3.3 ± 0.59 F2 0.025 ± 0.00550.0044% 67% 27% 9.2% 0.0044% 560 ± 40 8.9 ± 0.48  3.8 ± 0.44 F21 0.018 ±0.0063 0.013% 70% 27% 4.0% 0.056%  140 ± 7.6 4.0 ± 0.16 0.085 ± 0.037 F50.070 ± 0.011  0.010% 91% 6.4%  1.9% 0.41% 680 ± 49 3.0 ± 0.31 1.7 ± 1.2F8 0.048 ± 0.030  0.036% 67% 28% 5.0% 0.021%  130 ± 9.8 2.1 ± 0.20 1.4 ±1.2 Median 0.048 0.013% 68% 27% 5.0% 0.28% 540 5.0 1.7 SEM 0.023 0.0053%4.7%  3.4%  2.3% 0.092% 110 1.9  0.49

A well-recognized mechanism of mucosal immune defense is ‘immuneexclusion’ in which microorganisms in the gut are agglutinated bysecreted polyvalent IgA and IgM into clusters too large to diffusethrough mucus (Hamburger et al., Curr. Top. Microbiol. Immunol. 308:173(2006); Mantis et al., Mucosal Immunol. 4:603 (2011)). However, littleto no agglutinated HSV-1 was in these experiments, consistent withprevious findings that IgG is a relatively poor agglutinator (Berzofskyet al., In Fundamental Immunology. W. E. Paul, editor The Raven Press,New York, N.Y. 421 (1993)). Together, these observations suggest thatindividual HSV-1 virions in samples with elevated endogenous levels ofanti-HSV-1 IgG are slowed or trapped by multiple low-affinity bonds withCVM rather than by physical (steric) obstruction.

To confirm that trapping of HSV-1 in CVM was mediated specifically byIgG bound to virions and not by any other component in mucus that mightbe associated with elevated endogenous anti-HSV-1 IgG, HSV-1 specificIgG was affinity-purified from human intravenous immunoglobulin(starting with a pure clinical IgG preparation), and the purified IgGwas mixed into CVM samples that had low endogenous anti-HSV-1 IgG. Itwas found that addition of 1 μg/mL anti-HSV-1 IgG trapped HSV-1 with apotency comparable to that of endogenous anti-HSV-1 IgG (FIGS. 5A-5C;p<0.05 compared to native specimen without addition of anti-HSV-1 IgG).Lower anti-HSV-1 IgG doses were tested, and potent trapping of virionswas observed when ^(˜)333 ng/mL anti-HSV-1 IgG was added (p<0.05), andpartial trapping at 100 and 33 ng/mL anti-HSV-1 IgG added (both p<0.05).As controls, muco-adhesive PS remained markedly slowed or immobilizedand muco-inert PS-PEG freely diffusive in CVM samples treated with thehighest anti-HSV-1 IgG doses (FIG. 4B), confirming that the IgG did notcause HSV-1 trapping by altering mucus viscoelasticity or mesh spacing.Affinity-purified anti-HSV-1 IgG exhibited little neutralizing activityat 1 μg/mL and ^(˜)333 ng/mL (FIG. 5B), based on reduction of plaqueformation in Vero cells, suggesting that multiple low-affinity bondsbetween IgG and CVM can trap virions at IgG levels lower than thoseneeded to neutralize. HSV-1 was also trapped by a humanized monoclonalanti-gD IgG in CVM (FIG. 6A) but not by control, non-specific IgG (FIG.6A), underscoring the specificity of trapping via particularantibody-virus pairs, rather than a non-specific interaction oralteration of general mucus barrier properties. In good agreement withprevious studies (Olmsted et al., Biophys. J. 81:1930 (2001); Saltzmanet al., Biophys. J. 66:508 (1994)), both polyclonal anti-HSV-1 IgG andmonoclonal anti-gD IgG were only slightly slowed in CVM compared tosaline (FIG. 6B), suggesting both antibodies form only transient,low-affinity, bonds with CVM as individual molecules, yet facilitateeffective trapping of virions once they accumulate on the viral surfaceby forming low-affinity but polyvalent IgG-mucin bonds.

It was next sought to determine the biochemical basis of thelow-affinity bonds between IgG and CVM, The Fc domain of all IgGsharbors a conserved N-glycosylation site at Asn297, and many IgGeffector functions are Fe- and Asn297 glycan-dependent (Ha et al.,Glycobiology 21:1087 (2011)). Thus, F(ab′)₂ fragments (FIG. 7A) anddeglycosylated IgG (FIG. 7B) were prepared from the sameaffinity-purified anti-HSV-1 IgG to minimize any changes in HSV-1binding avidity (confirmed by ELISA), and the mobility of HSV-1pre-mixed with these modified analogs prior to addition to CVM (premixedto minimize interference by endogenous HSV-1 specific IgG) was measured.It was found that both F(ab′)₂ and deglycosylated IgG exhibitedsubstantially reduced trapping potency compared to intact IgG (FIG. 7C;p<0.05), suggesting that the low-affinity bonds IgG forms with mucinsare not only Fe-dependent, but also influenced by Fe glycosylation.

To determine whether trapping viruses in mucus can protect againstinfection in vivo, the ability of a non-neutralizing monoclonal IgG₁ toreduce HSV-2 transmission in the pH neutral mouse vagina was evaluated.This monoclonal IgG₁binds to the relatively sparse gG surfaceglycoprotein, and exhibited no neutralization activity across allconcentrations tested in vitro (FIG. 8A); mouse IgG₁ also possesseslittle to no complement (Ey et al., Mol. Immunol. 17:699 (1980);Michaelsen et al., Scand. J. Immunol. 59:34 (2004); Neuberger et al.,Eur. J Immunol, 11:1012 (1981)) and ADCC (Akiyama et al., Cancer Res.44:5127 (1984); Kipps et al., J Exp. Med. 161:1 (1985)) activity. Micewere challenged vaginally with 2 ID₅₀ HSV-2 with and without anti-gGIgG₁, and HSV infection was assayed by detection of virus shedding invaginal lavages three days post inoculation, a more sensitive assay ofinfection than visual observation of lesions, viral isolation fromsacral ganglia, or death (Zeitlin et al., Contraception 56:329 (1997)).Anti-gG IgG₁, at a concentration of 3.3 μg/mL and above, significantlyprotected against infection and reduced the average viral load comparedto either medium alone or control, non-specific IgG (FIGS. 8B and 8C,p<0.05). Interestingly, anti-gG IgG₁ appeared to only reduce the rate ofsuccessful vaginal HSV transmission; in mice that became infected, theextent of vaginal infection was comparable to that in mice receivingcontrol IgG, suggesting that the anti-gG IgG dosed did not eliciteffector functions that reduced the extent of virus spread in infectedmice compared to control IgG (FIG. 8C), Protection was also evaluated inmice that received a gentle vaginal wash to remove mucus withoutdetectable trauma to the epithelium (FIG. 9A). The removal of CVMincreased susceptibility to HSV-2 in control mice from ˜70% to ˜100%(FIG. 9B) but not the degree of HSV shedding in mice that becameinfected (FIG. 9C). This moderate (˜30%) increase in susceptibility islikely attributed to loss of innate protection by CVM itself: a CVMlayer prevents immediate direct contact between viruses and theepithelium, and contains factors, such as defensins, that may furthercontribute to overall reduction infectious HSV flux to the epithelium.More importantly, removal of CVM completely abolished the ˜50% extraprotection (from ^(˜)˜0% to ˜20% infection) afforded by anti-gG IgG₁ innative mice that cannot be attributed to innate immunity (FIG. 9B).Consistent with the hypothesis that trapping in mucus may facilitateprotection, these results together suggest much of the observedsynergistic enhancement in protection by anti-gG IgG₁ when CVM ispresent most likely occurred prior to HSV reaching target cells, ratherthan by immune mechanisms that can facilitate protection at the cellularlevel (e.g., complement or ADCC). These observations are also consistentwith the poor complement and ADCC activity of mouse IgG₁, as well asnumerous previous studies that have shown HSV can evade complement andother classical immune protective mechanisms (Brockman et al., Vaccine26 Suppl 8:194 (2008); Hook et al., J. Virol. 80:4038 (2008); Lubinskiet al., J. Exp. Med. 190:1637 (1999); Yuan et al., Nature Immunol. 7:835(2006)). Since even a non-neutralizing monoclonal IgG against arelatively sparse surface antigen can afford substantial protection,monoclonals against more abundant surface antigens, such as gD and gB,or those optimized to maximize interactions with mucus are likely toprovide even more potent protection at mucosal surfaces in vivo.

The first evidence of antibody-mucin affinity can be traced back morethan 30 years, when Kremer and Jager noted that infertility in humans isoften caused by anti-sperm antibodies (Jager et al., Fertil. Steril.36:792 (1981); Kremer et al., Fertil. Steril. 27:335 (1976)). Incervical mucus samples with high levels of anti-sperm Ab, they foundthat both individual and agglutinated sperm make no forward progress andshake in place for hours until they die, despite vigorous flagellarmotility. More recently, Phalipon et al. suggested secretory IgA canaggregate pathogenic Shigella flexneri in mouse nasal mucus secretionsvia the secretory component, anchoring the bacteria to the mucus gel andthereby ‘excluding’ them from infectious entry (Phalipon et al.,Immunity 17:107 (2002)).

In both of the above instances, the authors assumed that the antibodieswere attached firmly to the mucins. Similarly, the mucin-like Feybinding protein (FcγBP) has been proposed to serve an immunological rolein mucus through its ability to bind strongly to IgG Fc (Kobayashi etat, Gut 51:169 (2002)). Nevertheless, more recent evidence indicates theprimary function of FcγBP is instead to stabilize gastrointestinal mucusgel by covalently cross-linking Muc2 mucins (Johansson et at, Proc.Natl. Acad. Set. U.S.A. 108 Suppl 1:4659 (2011); Johansson et at, J.Proteome Res. 8:3549 (2009)). An IgG Fc-FcγBP-mucin crosslinkingmechanism also directly contradicts numerous prior efforts that havefailed to detect any significant binding of individual Ab to mucins(Clamp, Biochem. Soc. Trans. 5:1579 (1977); Cone, In Handbook of MucosalImmunology. P. L. Ogra et al., editors. Academic Press, San Diego,Calif. 43-64 (1999); Crowther et al., Fed. Proc. 44:691 (1985); Olmstedet al., Biophys. J. 81:1930 (2001); Saltzman et al., Biophys. J. 66:508(1994)). Indeed, previous FRAP experiments (Olmsted et al., Biophys. J.81:1930 (2001)) and those here demonstrate that IgG and other Ab diffuserapidly in human genital mucus, slowed only slightly compared to theirdiffusion in water. This can only be explained by weak and short-livedadhesive interactions between IgG and the mucin mesh, and not by thestrong binding of IgG by FcγBP (Kobayashi et al., J. Immunol. 143:2567(1989)). FcγBP also binds broadly to all IgG, and the interaction isthus subject to competitive inhibition (Kobayashi et at, J. Immunol.143:2567 (1989)). In the present experiments where exogenous HSV-1specific IgG was added to CVM, total levels of IgG already present inthe samples were hundreds- to thousands-fold higher than the HSV-1specific IgG doses added. Thus, for FcγBP to be responsible for theobserved trapping phenomena, FcγBP must have been present in greatermolar quantities than native IgG, which is unlikely given that theprotein has not been routinely identified in proteomic screens of humangenital tract fluid (see (Andersch-Bjorkman et al., Mol. Cell.Proteomics 6:708(2007)) vs (Dasari et al., J. Proteome Res. 6:1258(2007); Shaw et al., J. Proteome Res. 6:2859 (2007); Tang et at, J.Proteome Res. 6:2874 (2007)). In contrast to previous studies, byexamining the effect of IgG on virions in mucus gel rather than probingdirectly for interactions between individual IgG molecules and mucins,the present results document not only the potent trapping of individualvirions by multiple surface-bound IgG, but also that the IgG-mucininteractions are Fc- and glycan-dependent.

Trapping virions in genital tract mucus should markedly reduceheterosexual transmission of viral infections. Women acquire many of themajor sexually transmitted viral infections (e.g., HIV, HPV, and HSV) atrates on the order of 1 per 100 to 1,000 sex acts on average. Thissuggests few if any of these virions are able to infect target cells perintercourse, and therefore any reduction in the flux of virions thatreach target cells should proportionally reduce transmission rates.Blocking initial infection altogether, rather than attempting to clearinitial infections, may be especially critical for infections that aredifficult, if not impossible, to cure once established (e.g., HSV, HIV).In the recent gD2-AS04 HSV vaccine trial (Belshe et al., New Engl J.Med. 366:34 (2012)), protective efficacy was initially observed inseronegative women but not in men or seropositive women, and a largerstudy of seronegative women revealed only moderate efficacy againstHSV-1 (^(˜)35% efficacy against HSV-1 infection and 58% efficacy againstHSV-1 genital disease) but interestingly no protection against HSV-2. Inboth studies, the vaccine elicited neutralizing serum Ab against HSV aswell as HSV-specific cellular immune responses in all women and men.However, because mucosal levels of Ab were not monitored, it remainsunclear whether what little protection was observed could havecorrelated with mucosal Ab response. It is likely that generatingsufficient mucosal Ab levels remains a major bottleneck to an effectiveHSV vaccine. Inducing secreted Ab that bind to ‘non-neutralizing’surface epitopes should trap pathogens as effectively as those that bindto neutralizing epitopes, a prospect that may broaden potential antigentargets for vaccine development, especially against virions with rapidlyevolving neutralizing epitopes.

Example 3 Trapping of Salmonella typhimurium in Gastrointestinal Mucus

Fe-mediated trapping of pathogens in mucus, which directly blocksinfections at the portals of entry, may represent an exceptionallypotent mechanism by which the immune system can rapidly adapt andreinforce multiple mucosal surfaces against diverse and rapidly evolvingpathogens. A microscopy setup was developed that enables measuringbacterial mobility in real time directly in mucus overlaying intestinalepithelial tissue excised from mice (FIG. 10A). It was found thatSalmonella typhimurium can readily penetrate undiluted mucus secretionscoating the mouse gastrointestinal tract (duodenum, jejunum and ileum),but was effectively immobilized by topically applied anti-LPS andanti-flagella IgG (FIG. 10B). This immobilization occurred withoutinhibiting the flagella beating apparatus (Ab-coated Salmonella remainedhighly mobile in buffer) and without causing aggregation (i.e.,independent of the classical, aggregation-based mechanism of immuneexclusion (FIGS. 11A-11B). Furthermore, deglycosylated anti-LPSantibodies failed to trap the Salmonella, again highlighting thedependence of IgG-mucin interactions on Fc-N-glycans. These studieshighlight the ability for IgG-mucin interactions to function atdifferent mucosal surfaces, and act effectively on not just viralpathogens but also bacterial pathogens. The observed trapping in bothCVM (predominantly Muc5b mucins) and gastrointestinal mucus (Muc2mucins) suggests the molecular basis for Fe-mucin affinity is likelycommon among major secreted mucins—the long densely glycosylated fibersthat form mucus gels—and possibly mediated by glycans, since sugarsrepresent the major constituent of mucins (up to 80% by dry weight (Laiet al., Adv. Drug Deliv. Rev. 61:158 (2009))).

Example 4 Role of N-Glycans on IgG-Mucin Interactions

To further evaluate the role of N-glycans on IgG-mucin interactions, anumber of anti-gD antibodies (“HSV8”) with distinct glycosylationpatterns were tested in the CVM assay of Example 1. Antibodies withdistinct glycosylation profiles were produced in Nicotiana benthamianaplants using a viral-based transient expression system (magnICON).Transgenic Nicotiana benthamiana, in which plant-specific N-glycans(with core α1,3 fucose and β1,2 xylose) are reduced by RNAi inhibitionof plant-specific glycosyltransferases and specific glycosyltransferaseswere over-expressed (e.g., β1,4 galactosyltransferase, seewww.jbc.org/content/284/31/20479.full), were used as the host plant.Extract was clarified and IgG purified by protein A chromatography.Antibodies that were tested include HSV8-GnGn, in which contains about95% of the antibodies contain the core structure with GlcNAc, mannoseand terminal GlcNAc; HSV8-Gal, in which about 63% of the antibodies haveeither one or two terminal galactose sugar residues and about 5% havethe GnGn core structure; HSV8-Agly, which is free of any glycosylation;and HSV8 produced in 293 cell cultures, which have the general diversityin glycosylation profiles found in humans. Note that HSV8-Gal representsthe major serum IgG glycoform. We found that surprisingly, HSV8-Gal weresubstantially less potent at trapping viruses as HSV8-GnGn, whichsuggests antibodies that are engineered to have greater amount/extent ofGnGn glycosylation vs. the naturally found Gal-terminated glycosylationis expected to be more potent at trapping virions. This observation notonly further confirms the dependence on glycosylation, but underscoresthe importance of the precise nature of the glycosylation pattern.

As discussed above, the present invention is also based, in part, on thediscovery and characterization of weak binding interactions betweenantibodies and mucins and the ability of such antibodies to stop thepenetration of virions through mucus layers of the respiratory tract, asgenerally illustrated in FIG. 13. The antibody-mucin interaction can beused advantageously in methods for preventing and treating respiratoryinfection an entry of virions into a subject.

As mentioned above, the presently-disclosed subject matter includesantibodies, compositions, and methods for inhibiting and/or treatingviral infection, and/or eliminating virions from a mucosal surface ofthe respiratory tract. In particular, the presently-disclosed subjectmatter relates to antibodies and compositions capable of trappingvirions in mucus, thereby inhibiting transport of virions across orthrough mucus secretions.

One aspect of the invention relates to an antibody, e.g., a recombinantmonoclonal antibody molecule, comprising a human Fc portion and a set ofCDRs with specific affinity for a virion present in the lung of asubject.

In some embodiments, the antibody binds specifically to a single epitopeon a single virion. In some embodiments, the antibody is a multimericconstruct, e.g., wherein 2 or more Fabs (e.g., 2, 3, 4, 5, or more) areassociated with a single Fc domain. The Fabs may be the same ordifferent, and may recognize the same epitope on a virion, differentepitopes on the same virion, or epitopes on different virions. Themultimeric constructs are expected to facilitate more effectiveagglutination of both virus and/or virus-infected cells, resulting inmore effective elimination from mucosal surfaces. Such multimericantibody constructs can be prepared by linking multiple Fab domains viaa flexible peptide linker. The heavy- and light-chain gene sequences forIgG control antibodies and each of the Fab-based multimeric antibodiesmay first be optionally codon-optimized, then synthesized and clonedinto mammalian expression vectors (such as those offered by, e.g.,Integrated DNA Technologies). For each format, Fab-components may beseparated by a flexible peptide linker (e.g., amino acids comprisingabout 6 repeated units of GSSSS (SEQ ID NO:13), e.g., 2, 3, 4, 5, 6, 7,or 8 repeated units).

In some embodiments, the antibody may be a bispecific or multispecificconstruct against more than one virion. In one example, the constructmay bind both RSV and metapneumovirus (MPV). In some embodiments, abispecific antibody (e.g., Fab-IgG1, where an extra Fab is introduced inthe N-terminus immediately adjacent to the native Fab of IgG) can beproduced, e.g., by introducing separate orthogonal mutation sets (Lewiset al., Nature Biotechnol. 2014) into Fab A versus Fab B to ensureproper pairing of heavy and light chains. Fab A and Fab B may becombined with CH1/CL and Fc regions of human IgG1 Ab.

The CDRs may be any set of CDRs known in the art or later identifiedthat specifically bind a virion.

In some embodiments, the virus is Ebola virus and the CDRs are selectedfrom any combination of the CDRs found in known anti-Ebola virusantibodies.

In some embodiments, the virus is RSV and the CDRs are selected from anycombination of the CDRs found in known anti-RSV antibodies. In someembodiments, the CDRs are from any of the antibody sequences disclosedin U.S. Pat. No. 8,562,996 (referred to as palivizumab, or SYNAGIS),incorporated by referenced herein in its entirety, or derivativesthereof (e.g., motavizumab, Wu H et al, 2007, J. Mol. Biol. 368,652-665). In some embodiments, the CDRs may be one or more of the CDRsfound in any of the heavy and light chain sequences below.

Heavy Chain Variable Region: (SEQ ID NO: 1)QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTV SSLight Chain Variable Region: (SEQ ID NO. 2)DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTVHeavy Chain Variable Region:QVTLRESGPALVKPTQTLTLTCTFSGFSLS TSGMSVG WIRQPPGKALEWLA                                A                                 A                                ADIWWDDKKDYNPSLKS RLTISKDTSKNQVVLKVTNMDPADTATYYCAR         H      D        H      D SMITNWYFDV WGAGTTVTVSS (SEQ ID NO: 3)             Q         (SEQ ID NO: 4)     F       Q         (SEQ ID NO: 5)D  F F       Q         (SEQ ID NO: 6)D  F F       Q         (SEQ ID NO: 7) Light Chain Variable Region:DIQMTQSPSTLSASVGDRVTITC KCQLSVGYMH WYQQKPGKAPKLLIY                        SASS                         SASS                        SASSR                         SASSRDTSKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC FQGSGYPFT  F                                          F   FF DFGGGTKLEIK (SEQ ID NO: 8)       V    (SEQ ID NO: 9)      V    (SEQ ID NO: 10)       V    (SEQ ID NO: 11)      V    (SEQ ID NO: 12)

Any of the antibodies described herein may be muco-trapping antibodies,configured to transiently bind to mucus, as described above. Forexample, in some embodiments, the antibody comprises an oligosaccharideat a glycosylation site, the oligosaccharide comprising, consistingessentially of, or consisting of a pattern correlating with (providing)enhanced trapping potency of the antibody in mucus, and wherein theantibody specifically binds an epitope of a target virion. The uniqueglycosylation pattern/unique oligosaccharide component of the antibodyis designed to maximize trapping potency of the antibody once aplurality of antibodies are bound to the target virion, without undulyhindering the ability of the unbound antibody to diffuse readily throughmucus to rapidly bind a target virion. In certain embodiments, theantibody is one that exhibits a mobility in mucus that is reduced nomore than about 50%, e.g., no more than about 40%, 30, 20%, 10%, or 5%,relative to its mobility in solution (e.g., saline or water) andeffectively traps a target virion in mucus based on a plurality of boundantibodies (e.g., at least 50% of virions slowed by at least 90%). Insome embodiments, the antibody reduces the mobility of at least 50% ofthe virions, e.g., at least 60%, 70%, 80%, or 90% or more of the virionsby at least 90%, e.g., at least 95%, 96%, 97%, 98%, or 99% or more. Inother embodiments, the antibody reduces the percentage of virions thatcan penetrate mucus by at least 10%, e.g., at least 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more. Based on the disclosure herein, one ofskill in the art can readily identify/design oligosaccharide patternsthat provide the desired trapping potency. In other embodiments, theantibody has a sufficient binding rate to an epitope of the targetvirion to accumulate on the surface of the target virion at sufficientlevels to trap the target virion in mucus within one hour (e.g., within30 minutes or 15 minutes) at an antibody concentration in the mucus ofless than 5 μg/mL (e.g., less than 1 μg/mL or 0.1 μg/mL).

In some embodiments, the oligosaccharide component is bound to anN-linked glycosylation site in an Fc region of the antibody. TheN-linked glycosylation site can be an asparagine residue on the Fcregion of the antibody, for example, the Asn 297 asparagine residue. Theamino acid numbering is with respect to the standard amino acidstructure of a human IgG molecule.

As described in greater detail above, the N-glycan structure on humanIgG-Fc is typically dominated by a biantennary core structure thatshares a common core sugar sequence,Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser/Thr, with “antennae”initiated by N-acetylglucosaminyltransferases (GlcNAcTs) that helpattach additional sugars to the core. In IgG found in human serum, themost common structures are those that contain both N-acetylglucosamineon each branch with one terminal galactose (39%), two terminal galactose(20%), or one terminal galactose and one terminal sialic acid (15%).Together, antibodies that comprise at least one terminal galactoserepresents about 74% of the IgG-Fc glycoforms. A pure GnGn form (withterminal N-acetylglucosamine on each branch without terminal galactoseor sialic acid) may represent about 26% of the IgG-Fc glycoforms.

In some embodiments, the glycan does not contain any galactose residues.Without being limited by theory, it is believed that the presence ofgalactose compromises trapping potency. Antibodies with glycoforms thatdo not contain galactose represent just a small fraction of the entirerepertoire of glycoforms found in nature. The use of a population ofantibodies enriched with desirable glycoforms (whether naturallyoccurring or modified glycans) is advantageously used for trappingpathogens in mucus.

In some embodiments, the antibody of the invention is a mixture ofantibodies having different oligosaccharide components. In someembodiments, the mixture of antibodies comprises at least about 20%(e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, etc.) antibodies having the core glycanstructure described above (e.g., GnGn) with or without a fucose residue,e.g., at least about 40%, 50%, 60%, 70%, 80%, 90% or more.

In some embodiments, the mixture of antibodies is the mixture generatedin a human cell line, e.g., a 293 cell line, e.g., a 293T cell line. Insome embodiments, the mixture of antibodies is the mixture generated ina plant cell or in a plant.

The antibody may be useful for binding target virions to trap thepathogen in mucus of the respiratory tract to inhibit infection by thevirions. Target virions of the antibody can include any virus that caninfect a subject through a mucus membrane. Target virions furtherinclude synthetic systems comprising an antigen having an epitope, forexample particles or particulates (e.g., polystyrene beads) comprisingattached proteins, e.g., as might be used for bioterrorism.

Viruses include those that cause respiratory diseases, including,without limitation, influenza (including influenza A, B, and C); severeacute respiratory syndrome (SARS); respiratory syncytial virus (RSV);metapneumovirus; parainfluenza; adenovirus; human rhinovirus;coronavirus; and norovirus. Viruses also include those that do notnecessarily cause respiratory disease but can infect a subject by entrythrough the respiratory tract, e.g., Ebola virus.

Viruses include those that affect non-human animals, such as livestock,e.g., swine (e.g., porcine reproductive and respiratory syndrome virus(PRRSV), swine influenza, porcine circovirus), ruminants (e.g., RSV,adenovirus, reovirus), ungulates (e.g., bocaparvovirus), horses (e.g.,equine herpesvirus-1, equine herpesvirus-4, equine influenza virus),poultry (e.g., avian influenza virus, avian infectious bronchitis virus(IBV)), and the like.

The terms virus and viral pathogen are used interchangeably herein, andfurther refer to various strains of virus, e.g., influenza is inclusiveof new strains of influenza, which would be readily identifiable to oneof ordinary skill in the art.

In some embodiments, it is contemplated that an antibody according tothe presently-disclosed subject matter is capable of broadly binding toviruses containing lipid envelopes, which are not necessarily specificto one virus.

It was surprisingly discovered that sub-neutralization doses of anantibody can be used to effectively trap a target virion in mucus. Assuch, in some embodiments, wherein the antibody specifically binds aneutralizing epitope of the target virion, a sub-neutralization dose canbe used. A sub-neutralization doses is a dose below that which would beneeded to achieve effective neutralization.

As will be recognized by one of skill in the art, appropriate doses maydiffer between virions, between mucosal surfaces, and also betweenindividuals. It will also be recognized that different subjects anddifferent mucosal surfaces may have different optimal glycan patternsand optimal antibody-mucin affinities, contributing to different optimaldoses.

It is further proposed herein that antibodies that selectively bindnon-neutralizing epitopes of a target virion can be used to effectivelytrap the target virion in mucus. As such, in some embodiments, theantibody specifically binds a non-neutralizing epitope, e.g., one ormore non-neutralizing epitopes.

The presently-disclosed subject matter further includes an antibody thatselectively binds a conserved epitope of a target virion. A benefit oftargeting a conserved epitope would be to preserve efficacy of theantibody as against new strains of the virus. Targeting such epitopeshas been avoided at times in the past because they were viewed as beingineffective targets; however, in view of the disclosure herein thatnon-neutralizing epitopes can serve as effective targets and/or thatsub-neutralization doses can be effective for inhibiting infection,previously dismissed conserved epitopes of target virions can be seen aseffective targets.

As noted above, it was determined that the low-affinity bindinginteractions that an antibody forms with mucins are influenced byantibody glycosylation, and are also Fc-dependent. As such, thepresently-disclosed subject matter includes antibodies having apreserved and/or engineered Fc region. Such antibodies can be, forexample, one or more of IgG, IgA, IgM, IgD, or IgE. In certainembodiments, the antibodies are IgG. In some embodiments, the antibodiesare one or more subclasses of IgG, e.g., IgG1, IgG2, IgG3, IgG4, or anycombination thereof.

In some embodiments, the antibody has a sufficient binding rate and/orbinding affinity to an epitope of the target virion to accumulate on thesurface of the virion at sufficient levels to trap the virion within onehour after administration of the antibody at an antibody concentrationof less than about 5 μg/mL, although excess antibody may be used (e.g.,about 5 μg/mL or more, 10 μg/mL or more, 15 μg/mL or more, 20 μg/mL ormore, 25 μg/mL or more, 30 μg/mL or more, 40 μg/mL or more, 50 μg/mL ormore, 60 μg/mL or more, 70 μg/mL or more, 80 μg/mL or more, 90 μg/mL ormore, 100 μg/mL or more, etc.). The term “trap” in this instance refersto reduction of further movement through the mucus. In some embodiments,the target virion may be trapped within about 30 minutes, e.g., about25, 20, 15, or 10 minutes after administration of the antibody. In someembodiments, the antibody traps the target virion at an antibodyconcentration of less than about 4, 3, 2, or 1 μg/mL.

As mentioned, formulations for administration, including intranasaladministration, etc., are contemplated for use in connection with thepresently-disclosed subject matter. All formulations, devices, andmethods known to one of skill in the art which are appropriate fordelivering the antibody or composition containing the antibody to one ormore mucus membranes of the respiratory tract of a subject can be usedin connection with the presently-disclosed subject matter. The antibodycan be formulated for nasal administration or otherwise administered tothe lungs of a subject by any suitable means, e.g., administered by anaerosol suspension of respirable particles comprising the antibody,which the subject inhales. The respirable particles can be liquid orsolid. The term “aerosol” includes any gas-borne suspended phase, whichis capable of being inhaled into the bronchioles or nasal passages.Specifically, aerosol includes a gas-borne suspension of droplets, ascan be produced in a metered dose inhaler or nebulizer, or in a mistsprayer. Aerosol also includes a dry powder composition suspended in airor other carrier gas, which can be delivered by insufflation from aninhaler device, for example. See Ganderton & Jones, Drug Delivery to theRespiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviewsin Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J.Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particlescomprising the antibody can be produced by any suitable means, such aswith a vibrating mesh nebulizer, a pressure-driven aerosol nebulizer, oran ultrasonic nebulizer, as is known to those of skill in the art. See,e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprisingthe antibody can likewise be produced with any solid particulatemedicament aerosol generator, by techniques known in the pharmaceuticalart. In some embodiments, the nebulizer and nebulization conditions areselected to produce aerosol particles of a desired size. In oneembodiment, the nebulized composition has a Mass Median AerodynamicDiameter (MMAD) in the 2-5 mm range, as measured using a Next GenerationImpactor.

In some embodiments of the presently-disclosed subject matter, acomposition includes a first antibody and a second antibody, asdisclosed herein, wherein the first antibody specifically binds a firstepitope of the target virion and the second antibody specifically bindsa second epitope of the target virion, wherein said first epitope isdistinct from the second epitope. In certain embodiments, thecomposition includes three or more different antibodies, e.g., 3, 4, 5,6, 7, 8, 9, 10, or more different antibodies, wherein each antibodyspecifically binds a different epitope of the target virion.

It may also be desirable to provide a composition that can providetreatment or prevention of infection due to more than one target virus.In some embodiments of the presently-disclosed subject matter, acomposition includes a first antibody and a second antibody, asdisclosed herein, wherein the first antibody specifically binds anepitope of a first target virion and the second antibody specificallybinds an epitope of second target virion. In certain embodiments, thecomposition includes three or more different antibodies, e.g., 3, 4, 5,6, 7, 8, 9, 10, or more different antibodies, wherein each antibodyspecifically binds an epitope of a different target virion.

In some embodiments, the pharmaceutical composition can further includean additional active agent, e.g., a prophylactic or therapeutic agent.For example, the additional active agent can be an antimicrobial agent,as would be known to one of skill in the art. The antimicrobial agentmay be active against algae, bacteria, fungi, parasites (helminths,protozoa), viruses, and subviral agents. Accordingly, the antimicrobialagent may be an antibacterial, antifungal, antiviral, antiparasitic, orantiprotozoal agent. The antimicrobial agent is preferably activeagainst infectious diseases. Examples of suitable antimicrobial agents(e.g., one or more antibacterial, antifungal, antiviral, antiparasitic,or antiprotozoal agents) include those described above.

The presently-disclosed subject matter further includes methods oftreating, inhibiting, or preventing a viral infection in a subject inneed thereof, the viral infection characterized by a virion in the lungof the subject, the method comprising administering, via an inhaledroute, a recombinant monoclonal antibody molecule comprising a human Fcportion and a set of CDRs with specific affinity for the virion, therebytreating, inhibiting, or preventing the infection. The recombinantmonoclonal antibody molecule can be an antibody and/or composition asdisclosed herein. In certain embodiments, the methods compriseadditional steps such as one or more of isolating the antibodies,preparing a composition of the isolated antibodies, determining thelevel of antibodies in the mucus of the subject before administering theantibodies, and determining the level of antibodies in the mucus of thesubject after administering the antibodies.

The virion may be any virus as discussed herein. In some embodiments,the virion is Ebola virus. In some embodiments, the virion is RSV. Insome embodiments, the antibody comprises any of the CDR sequencesdisclosed herein.

The antibodies and compositions of the present invention according tothe methods described herein are administered or otherwise applied bydelivering the composition, e.g., to the lungs, e.g., by inhalation. Thesubject may be one where an infection is already present in the lungs(an actual site of infection) or where an infection is likely to occurin the lungs (a potential site of site of infection in an uninfectedindividual). In some embodiments, the antibodies and compositions may bedelivered to the respiratory tract, e.g., the nasal cavity and thelungs, by any method known in the art to be effective. In someembodiments, the antibodies and compositions may be delivered directlyto the respiratory tract. In other embodiments, the antibodies andcompositions may be systemically delivered such that the antibodies aresecreted into the mucus of the subject. Accordingly, the compositions asdescribed above may be delivered to a mucosal surface of the respiratorytract.

In some embodiments, the antibody is formulated for delivery to therespiratory tract. In some embodiments, the molecule is formulated as anaerosol composition. In some embodiments, the aerosol composition issuitable for nebulization. Any type of suitable nebulizer may be used.In one embodiment, the aerosol composition is nebulized by a vibratingmesh nebulizer. In some embodiments, the nebulizer and nebulizationconditions are selected to produce aerosol particles of a desired size.In one embodiment, the nebulized composition has a Mass MedianAerodynamic Diameter (MMAD) in the 2-5 mm range, as measured using aNext Generation Impactor.

An effective amount of the antibody can be administered. As used herein,an “effective amount” of the antibody for inhibition or prevention ofinfection refers to a dosage sufficient to inhibit or prevent infectionby the target virion. As used herein, an “effective amount” of theantibody for treatment of infection refers to a dosage sufficient toinhibit spread of the target virion from infected cells to non-infectedcells in the subject and/or to inhibit spread of the target virion fromthe infected subject to another subject, e.g., a non-infected subject.The effective amount can be an amount sufficient to trap an amount ofthe target virion in mucus. As will be recognized by one of skill in theart, the amount can vary depending on the patient and the target virion.The exact amount that is required will vary from subject to subject,depending on the species, age, and general condition of the subject, theparticular carrier or adjuvant being used, mode of administration, andthe like. As such, the effective amount will vary based on theparticular circumstances, and an appropriate effective amount can bedetermined in a particular case by one of skill in the art using onlyroutine experimentation. In some instances, an effective amount of theantibody that specifically binds the target virion can be an amount thatachieves a concentration of the antibody in the mucus of about 50 ng/mLto about 1000 μg/mL, e.g., about 0.1 μg/mL to about 100 μg/mL, about 0.5μg/mL to about 100 μg/mL, about 1 μg/mL to about 50 μg/mL, about 5 μg/mLto about 500 μg/mL, about 5 μg/mL to about 300 μg/mL, about 20 μg/mL toabout 200 μg/mL, or any range therein (e.g., between 0.5 μg/mL to about20 μg/mL).

In some embodiments, the antibody may be administered in two or morestages with different doses in each stage. For example, higher doses canbe administered initially in order to clear target virions that arepresent in the mucus of exposed or infected subjects and ensure thatsufficient amounts of antibody remain in the mucus to provideprotection, e.g., for about 24 hours. In later stages, lower doses canbe administered to maintain protective levels of the antibody. In otherembodiments, protective doses can be administered to subjects that arelikely to be exposed to a virus and higher doses can be administered ifinfection occurs.

In some embodiments, the antibody or composition is administered atregular intervals until an effect is achieved. The interval may be e.g.,multiple times a day (e.g., every 1, 2, 3, 4, 5, 6, 8, 12, 18 or 24hours, e.g., every 3-24 hours)), once every 24, 48, or 72 hours, or oncea week.

As will be recognized by one of skill in the art, the term “inhibiting”or “inhibition” or “preventing” or “prevention” does not refer to theability to completely eliminate the possibility of infection in allcases. Rather, the skilled artisan will understand that the term“inhibiting” or “preventing” refers to reducing the chances of virionsmoving through mucus beyond the mucus membrane such that infection of asubject can occur, such as reducing chances of infection by a virionwhen such virion is bound to trapping antibodies in mucus. Such decreasein infection potential can be determined relative to a control that hasnot been administered the antibodies of the invention. In someembodiments, the decrease of inhibition potential relative to a controlcan be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.

In some embodiments inhibiting or treating an infection in a subject cancomprise trapping a virion in mucus. As such, in some embodiments amethod of trapping a target virion in mucus is provided, which methodincludes administering to a mucosa of the respiratory tract of thesubject an antibody or composition as described herein.

In some embodiments, a method of inhibiting or treating an infection ina subject, and/or trapping a virion in the mucus of a subject, involvesadministering to a mucosa of the respiratory tract of the subject acomposition comprising an isolated antibody that specifically binds anon-neutralizing epitope of a target virion. The antibody can be anon-neutralizing antibody. In some embodiments, the non-neutralizingantibody is provided at a concentration above a predetermined amount.

In some embodiments, a method of inhibiting or treating an infection ina subject, and/or trapping a virion in the mucus of a subject, involvesadministering to a mucosa of the respiratory tract of the subject acomposition comprising an isolated antibody that specifically binds aneutralizing epitope of a target virion, wherein the antibody isprovided at a sub-neutralization dose.

Having described the present invention, the same will be explained ingreater detail in the following, examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 5 Blocking Penetration of Ebola Viruses in Human Airway Mucus

Ebola virus, a member of the filovirus family of viruses that causesevere and often fatal hemorrhagic fevers, readily infects many celltypes, including immune cells, fibroblasts, endothelial cells andepithelial cells. Given the broad tissue tropism of Ebola, effectivetreatments of systemic Ebola infection generally require high doses oftherapeutic molecules administered systemically. For instance, ZMapp™, acocktail of three chimeric monoclonal antibodies, was evaluated at 50mg/kg in a randomized, controlled clinical trial in Guinea, Sierra Leoneand Liberia during the 2014-2016 Ebola outbreak.

Rather than treating Ebola infections systemically, a potentialalternative strategy is to block or treat infections at the portals ofentry before virions proliferate and spread throughout the body. Inaddition to transmission by direct contact with the blood, bodilyfluids, or skin of Ebola-positive individuals, it is possible that Ebolamay be transmitted via virus-laden droplets generated from a heavilyinfected individual by coughing, sneezing, vomiting or medicalprocedures that are directly propelled onto the mucus membranes of anearby person. The term droplet-based aerosol transmission may be usedto differentiate this potential mechanism from strict airbornetransmission of individual viruses, which is generally considered anunlikely mechanism of Ebola transmission. While aerosol transmission ofEbola has not been ascertained in humans, it has been demonstrated inmultiple animal studies, including with non-human primates. Given thepossibility of aerosol transmission of Ebola to humans, as well as thepotential threat of aerosolized filovirus-based biowarfare agents, wesought to investigate the fate of Ebola deposited at mucosal surfaces.

Mucus membranes are characterized by a layer of mucus secretions thatcan trap diverse foreign particles and pathogens, facilitate theirelimination via natural mucus clearance mechanisms, and consequentlyreduce the flux of pathogens reaching target cells. Human airway mucus(AM) is likely responsible in part for the relatively modesttransmission rates of many respiratory viruses, but it is also likelythat AM can be reinforced to further limit the flux of pathogensreaching the underlying epithelium. As described above, IgG antibodies(Ab) in cervicovaginal mucus can trap viruses in mucus via multiplelow-affinity Fc-mucin bonds between IgG accumulated on the virus surfaceand mucins, akin to a Velcro® patch. Further, the immobilization of H1N1and H3N2 influenza viruses in human AM may be correlated to the presenceof influenza-binding IgG and IgA. Here, we investigate whether topicallydosed IgG against Ebola may similarly trap Ebola in AM and facilitatetheir elimination from the airways.

Ebola virus-like particles (VLP) were prepared by transfecting 293Tcells with plasmids encoding Gag-mCherry and Ebola GP. 293T cells weremaintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich,St. Louis, Mo.) supplemented with 10% fetal calf serum (FBS) and 2 mML-glutamine (DMEM-10). All cell cultures were maintained at 37° C. in ahumidified 5% CO₂ atmosphere. 293T cells (2.0×10⁶) were seeded in a 25cm² flask (Thermo Scientific, Rochester, N.Y.) and transfected with theexpression plasmids using X-tremeGENE HP DNA Transfection Reagent (RocheDiagnostics, Indianapolis, Ind.). Gag-mCherry and Ebola GP plasmids weremixed in a 1:1 ratio (1.5 μg of each), and added to 500 μL of serum freeDMEM with 9 μL of X-tremeGENE HP DNA Transfection Reagent. The mixturewas incubated at room temperature for 30 min before being added to theculture of 293T cells. After 3 to 5 hr incubation at 37° C. in 5% CO₂,transfected cells were washed extensively with DMEM and incubated foradditional 24-48 hr with 2 mL of DMEM-10 at 37° C. in 5% CO₂.Supernatants from virus particle-producing cultures were then collectedand clarified by centrifugation for 10 min at 300×g, filtered through alow protein binding 0.45 μM syringe filter (Millipore, Bedford, Mass.)and partially purified through 25% w/v sucrose in Hepes-NaCl buffer bycentrifugation at 221,630×g at 4° C. for 2.5 hr. The pellet wasresuspended overnight at 4° C. in 10% sucrose in Hepes-NaCl buffer,aliquoted, and stored at −80° C.

To measure incorporation of Ebola GP protein into the VLP, sucrosegradient-purified VLP were lysed in buffer containing 100 mM Tris-HCl(pH 8.0), 100 mM NaCl, 2% Triton X-100 and protease inhibitors at 4° C.for 30 min. Recombinant Ebola glycoprotein rGPd™ (#0501-001; IBTBioservices, Rockville, Md.) was run as a positive control. Samples wereboiled in SDS-PAGE sample buffer with 2-mercaptoethanol and separated on4%-12% Tris-Glycine gradient gels (Invitrogen, Grand Island, N.Y.),transferred to nitrocellulose membranes, and probed with Mouse EbolaGPII Monoclonal Antibody (MyBiosource, San Diego, Calif.) at aconcentration of 1:2,000, as shown in FIG. 14A. Specificity of ZMapp™binding to Ebola VLP was demonstrated via immunoprecipitation. Ebola VLPwere incubated with ZMapp™, individual Ebola-binding IgG, or α-Biotin at4° C. for 3 hr. The antigen-antibody complex was pulled down usingProtein G beads, and Ebola GP was probed as described above, and shownin FIG. 14B. The hydrodynamic diameter of Ebola VLP was measured using aNanoSight NS500 (Malvern Instruments, Malvern, UK). Samples were dilutedto a concentration of ˜10⁸ particles/mL in 20 nm filtered PBS, and five60 second videos were taken of each sample.

Both the ZMapp™ and any of the individual Ebola-binding mAbs used hereinwere modified to enhance musical binding, by enriching the population ofEbola-binding mAbs to include a majority (e.g., 50% or more, 55% ormore, 60% or more, 65% or more, 70% or more 75% or more; 80% or more 85%or more, etc. such as 85%-95% etc.) having a G0 glycosylation pattern onthe Fc region (e.g., a glycosylation pattern comprising the biantennarycore glycan structure Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 withterminal N-acetylglucosamine on each branch). Native anti-Ebola mAb(e.g., human anti-Ebola mAb) has a much lower G0 content.

Fluorescent, carboxyl-modified polystyrene beads (PS-COOH) and PEGylatednanoparticles (PS-PEG) sized approximately 100 nm were prepared andcharacterized. Fluorescent, carboxyl-modified polystyrene beads(PS-COOH) sized 100 nm were purchased from Molecular Probes (Eugene,Oreg.). PEGylated nanoparticles (PS-PEG) were prepared by conjugating 2kDa amine-modified PEG (Rapp Polymere, Tuebingen, Germany) to PS-COOHparticles via a carboxyl-amine reaction. Particle size and -potentialwere determined by dynamic light scattering and laser Doppleranemometry, respectively, using a Zetasizer Nano ZS (MalvernInstruments, Southborough, Mass.). Size measurements were performed at25° C. at a scattering angle of 90°. Samples were diluted in 10 mM NaClsolution, and measurements were performed according to instrumentinstructions. PEG conjugation was confirmed by a near-neutralζ-potential (Table 3).

TABLE 3 Characterization of PEG-modified nanoparticles. Size andζ-potential values for carboxyl-modified beads are provided forcomparison. Surface Diameter ζ-potential Size (nm)* Chemistry (nm) (mV)100 COOH 109 ± 4 −55 ± 5 100 PEG 132 ± 3  −7 ± 3 *Provided by themanufacturer.

Fresh human airway mucus (AM) was obtained from healthy adult patients(Table 4) intubated for general anesthesia during elective surgery,following a protocol that was deemed non-human subjects research by theUniversity of North Carolina at Chapel Hill Institutional Review Board.After surgery, the endotracheal tube was removed from the patient, andmucus coating the tube was collected by gentle centrifugation. AM thatwas non-uniform in color or consistency or had visible bloodcontamination was discarded. Samples were treated with proteaseinhibitor immediately after collection to minimize potential enzymaticdegradation, and stored at 4° C. until microscopy, typically within 24hr.

TABLE 4 Demographic information for donors of airway mucus specimens.Donor ID Gender Age Smoker Status AM01 F 28 N AM02 M 39 N AM03 M 22 YAM04 F 48 Y AM05 F 59 N AM06 F 66 N AM07 F 66 N AM08 F 61 N AM09 M 26 NAM10 F 62 N AM11 M 88 Y AM12 M 55 N

Total immunoglobulin levels in AM were quantified by conventional ELISAand confirmed using the Human Isotyping Kit on Luminex (HGAMMAG-301K;Millipore, Billerica, Mass.) according to manufacturer protocol.Briefly, 20× stock isotyping beads were vortexed, sonicated, diluted to1×, and incubated with 50 μL of serially diluted AM supernatant at 1:2beads:AM volume ratio. After 1 hr, the beads were separated from AMsupernatant using a magnetic plate, and washed twice with wash buffer.The beads were then incubated with 25 μL of 1× anti-Human Kappa andLambda-PE for 1 hr, washed twice, and resuspended in Luminex DriveFluid. Fluorescence intensities indicative of immunoglobulin levelspresent in AM were measured using the Luminex MAGPIX system, and dataanalysis was performed using Milliplex Analyst (v3.5.5.0; Vigene TechInc., Carlisle, Mass.). All incubations were carried out at roomtemperature in the dark with vigorous agitation. Total Ig and IgGisotypes levels are shown in (Table 5).

TABLE 5 Characterization of immunoglobulin levels in airway mucus. DonorTotal IgG Total IgA Total IgM ID IgG₁ IgG₂ IgG₃ IgG₄ (μg/mL) (μg/mL)(μg/mL) AM01 57.4% 33.0% 8.3% 1.2% 559 730 107 AM02 69.8% 23.6% 6.6%0.0% 3,171 360 90 AM03 11.0% 78.9% 7.6% 2.4% 109 28 14 AM04 52.3% 45.2%2.1% 0.4% 1,481 196 48 AM05 9.8% 88.0% 1.6% 0.6% 6,138 123 465 AM0645.8% 44.4% 7.3% 2.4% 3,788 645 657 AM07 45.4% 47.9% 2.3% 4.3% 3,9271,005 416 AM08 25.3% 67.9% 5.0% 1.8% 1,951 290 452 AM09 24.6% 65.9% 5.7%3.8% 158 36 25 AM10 16.9% 73.8% 8.2% 1.1% 387 127 36 AM11 57.3% 33.6%7.0% 2.1% 110 339 24 AM12 39.9% 51.6% 8.4% 0.2% 481 602 115 Median 42.6%49.7% 6.8% 1.5% 1,020 315 99 SEM 5.8% 5.8% 0.7% 0.4% 550 85 63

Dilute particle solutions (˜10⁸-10⁹ particles/mL, 1 μL) and different Ab(2 μL, to a final concentration of 22 μg/mL) were added to 20 μL offresh, undiluted AM in custom-made chambers, and samples were incubated1 hr at 37° C. before microscopy for Multiple particle trackinganalysis. All conditions were tested in aliquots of the same AM samples,allowing direct comparison between conditions. The trajectories of thefluorescent particles in AM were recorded using an EMCCD camera (Evolve512; Photometrics, Tucson, Ariz.) mounted on an inverted epifluorescencemicroscope (AxioObserver D1; Zeiss, Thornwood, N.Y.), equipped with anAlpha Plan-Apo 100×/1.46 NA objective, environmental (temperature andCO₂) control chamber and an LED light source (Lumencor Light EngineDAPI/GFP/543/623/690). Videos (512×512, 16-bit image depth) werecaptured with MetaMorph imaging software (Molecular Devices, Sunnyvale,Calif.) at a temporal resolution of 66.7 ms and spatial resolution of 10nm (nominal pixel resolution 0.156 μm/pixel). The tracking resolutionwas determined by tracking the displacements of particles immobilizedwith a strong adhesive. Particle trajectories were analyzed using VideoSpotTracker (University of North Carolina, Chapel Hill, N.C.). Sub-pixeltracking resolution was achieved by determining the precise location ofthe particle centroid by light-intensity-weighted averaging ofneighboring pixels. Trajectories of n≥130 particles per frame on average(typically corresponding to n≥300 total traces) were analyzed for eachexperiment, and 8-9 independent experiments for each condition wereperformed in AM collected from unique subjects. The coordinates ofparticle centroids were transformed into time-averaged mean squareddisplacements (MSD), calculated as<Δr²(τ)>=[x(t+τ)−x(t)]²+[y(t+τ)−y(t)]² (where τ=time scale or time lag),from which distributions of MSDs and D_(eff) were calculated. Mobileparticles were defined as those moving more than approximately 200 nm(i.e. roughly twice the particle diameter) within 0.2667 s.

MSD may also be expressed as MSD=4D₀τ^(α), where α, the slope of thecurve on a log-log scale, is a measure of the extent of impediment toparticle diffusion (α=1 for pure unobstructed Brownian diffusion; α<1indicates increasing impediment to particle movement as a decreases). Alimitation with 15D particle tracking is the inability to track rapidlydiffusing species due to diffusion out of the focal plane, particularlywith sub 200 nm species. Consequently, MSD calculations at longer timescales will underestimate the true mobility of these species. Therefore,a was calculated only over the linear portion of the log-log MSD curve(here, up to τ=0.6 s). Calculating a to span τ=1 s results in a value of0.71 vs. 0.80 for Ebola VLP in AM with no Ab, but had negligible impacton a values for Ebola VLP in AM treated with ZMapp™ or individual mAb.

In order to predict particle population behavior, we performed a firstpassage time analysis, calculating the expected time for 10% and 50% ofa particle population to pass through a 50 μm thick layer of mucus.Given the diffusivity D of a particle, the probability that the particlehas not passed through a layer of thickness L as of a given time t maybe described by an explicit “survival function”. Using T to denote thetime it takes for the particle to pass through the layer, the formulafor this “survival function” is

${P( {T > t} \middle| D )} = {\frac{4}{\pi}{\sum\limits_{k = 0}^{\infty}{\frac{( {- 1} )^{k}}{{2k} + 1}\exp \; {( \frac{{- ( {{2k} + 1} )^{2}}\pi^{2}Dt}{4L^{2}} ).}}}}$

Suppose that a heterogeneous population of particles have individualdiffusivities {D_(i)} with respective weights {w_(i)} where i rangesfrom 1 to the number of particles N. The expected fraction of particlesthat remain in the fluid layer as of time t is then equivalent to thefollowing weighted survival function:

${P( {T > t} )} = {\frac{\Sigma_{i = 1}^{N}{P( {T > t} \middle| D_{i} )}w_{i}}{\Sigma_{i = 1}^{N}w_{i}}.}$

We set w_(i) to be the number of frames in which the i^(th) particle ispresent. For each weighting, we calculated the times t₁₀ and t₅₀ forwhich P(T>t₁₀)=0.9 and P(T>t₅₀)=0.5.

A slightly hypotonic buffer, prepared by diluting Gibco® 1×PBS with anequal volume of Milli-Q® water to ˜150 mOsm/kg, was used to deliverEbola VLP to the mouse lung (hypotonic solutions were found to promoteadvective flow to the epithelium, allowing us to more rigorouslyevaluate whether ZMapp™ Ab can prevent Ebola from crossing the AMlayer). ZMapp™ cocktail (c2G4, c116C6FR1 and c4G7 mixed at a 1:1:1ratio, total 25 μL per mouse) or hypotonic PBS was first loaded into aPenn Century Microsprayer (FMJ-250; Penn Century, Inc., Wyndmoor, Pa.),aerosolized and delivered to the airways of Balb/c mice (female, 8-10weeks) anesthetized in an isoflurane chamber. After 15 min, Ebola VLPprepared in hypotonic PBS were instilled using the microsprayer at 25 μLper mouse, for both ZMapp™-treated and control mice. A third set of micewere treated with two instillations of hypotonic PBS to measure theautofluorescence of mouse lung tissue. Experimental and control groupshad n=3 mice each. All experimental protocols were approved by theUniversity of North Carolina at Chapel Hill Institutional Animal Careand Use Committee, and conform to the Declaration of Helsinkiconventions for the use and care of animals.

To investigate the distribution of Ebola VLP, mice were euthanized 30min after the final microsprayer instillation and the entire lung,including trachea, was dissected. The dissected lung was kept intact,washed with 1×PBS, and embedded in 100% optimal cutting temperature(OCT) compound before freezing at −80° C. After overnight freezing, 10μM thick transverse cross sections of the upper airways were obtainedvia cryosectioning at −20° C. and stained with DAPI. Cryosections wereimaged using an Olympus FV1000 MPE laser scanning confocal microscope(Olympus Life Science Solutions, Center Valley, Pa.) at 20×magnification and the following excitation/emission spectrum: TRITC(559/603 nm) and DAPI (405/422 nm). Fluorescence intensity wasquantified using ImageJ (National Institutes of Health, Bethesda, Md.)for on average 10 cross sections per mouse, and background fluorescencewas subtracted.

Data averages are presented as means with standard error of the mean(SEM) indicated. Statistical comparisons were limited to two groups. Aone-tailed, paired Student's t-test was used for all comparisons, sincedifferent conditions were tested in aliquots of the same mucus samples.Differences were deemed significant at an alpha level of 0.05.

The Ebola virus-like particles were found to quickly penetrate humanairway mucus. Wild-type Ebola virus requires Biosafety Level 4containment that few laboratories have access to. Therefore, toinvestigate the fate of Ebola in mucus, we prepared fluorescent,non-infectious Ebola VLP comprised of HIV-1 Gag-mCherry capsid proteinsin the core and the Zaire glycoprotein (GP), from the same species as inthe West Africa epidemic in 2014-2016, on the surface. The same strategywas previously used to prepare both HIV and influenza VLP. GPincorporation into Ebola VLP was confirmed via Western blot (FIG. 14A),and dynamic light scattering showed that the VLP possessed ahydrodynamic diameter of 102±3 nm. All three IgGs of the ZMapp™ cocktailbound specifically to the VLP, confirming the presence ofstructurally-intact GP (FIG. 14B).

To avoid the effects of dilution with using hypertonic saline to inducesputum expectoration, we obtained undiluted human airway mucus directlyfrom freshly extubated endotracheal tubes. Ebola VLP were readilydiffusive in all AM secretions tested, as shown in FIG. 15A, exhibitingdiffusive motion comparable to that of similarly-sized, polyethyleneglycol-coated polystyrene (PS-PEG) nanoparticles engineered to evadeadhesion to mucins and penetrate various mucus secretions. In the sameAM secretions, similarly sized carboxyl-modified polystyrene (PS-COOH)nanoparticles that are muco-adhesive were extensively immobilized,confirming that the rapid diffusivity observed for Ebola VLP was not dueto a degraded mucin matrix. Nearly all Ebola VLP (>75% in all samples,on average ˜90%) possessed diffusivities in excess of approximately 200nm, or twice the particle diameter, at a time scale of 0.2667 s, asshown in FIG. 15B. The geometrically averaged ensemble mean squareddisplacement (<MSD>) of Ebola was only ˜10-fold reduced compared totheir theoretical speeds in buffer (FIG. 15C; at a time scale of 0.2667s), with a slope a of 0.80 for the log <MSD> vs. log time scale plot(α=1 for pure unobstructed Brownian diffusion, e.g., particles in water,and a becomes smaller and approaches zero as obstruction to Browniandiffusion increases). The geometrically averaged effective diffusivity(<D_(eff)>) for Ebola VLP was 0.43 μm²/s, about 150-fold higher thanthat of PS-COOH nanoparticles (FIG. 15D).

Along the major conducting airways, the approximate thickness of mucuslining the bronchial airways is ˜50 μm, and the entire mucus blanket canbe renewed in as little as 15-30 minutes. Therefore, we performed afirst passage time estimate, using the measured diffusivities ofindividual VLP in AM to determine the time needed for viruses to diffuseacross a 50 μm thick AM layer. We found that upon airway deposition,nearly 10% of Ebola virions can penetrate the luminal AM layer withinapproximately 5 minutes, and nearly 50% can penetrate the AM layer in aslittle as 30 minutes (FIG. 17B). These results suggest limiting rapidEbola penetration of AM may be an important strategy to reduce Ebolainfection by decreasing the flux of virions reaching the underlyingairway epithelium.

The treatment of human airway mucus with ZMapp™ may immobilize Ebolavirus-like particles. While total Ig and IgG isotype levels variedsubstantially across AM samples (Table 5), the rapid diffusion of EbolaVLP in AM was remarkably consistent, suggesting endogenous antibody didnot impact Ebola VLP mobility. We next evaluated whether Ebola-bindingIgG, in the form of the three chimeric monoclonal antibody (mAb)cocktail ZMapp™, could enhance the diffusional barrier properties of AMagainst Ebola. The addition of modest levels of Ebola-binding IgG intoAM effectively reduced the mobility of Ebola VLP, with the majority ofVLP moving much less than their diameters over at least 20 s (FIG. 16A).Indeed, the <D_(eff)> of Ebola VLP in ZMapp™-treated AM decreased by˜27-fold compared to <D_(eff)> in the same native AM secretions withoutAb (FIG. 17A). Likewise, the fraction of mobile VLP was reduced from 90%to 29% (FIG. 16B), while <MSD> was over 260-fold lower than theoreticalVLP speeds in buffer (FIG. 16C; at a time scale of 0.2667 s). Theincreased hindrance to rapid diffusion is also evident from the log<MSD> vs. log time scale slope a of 0.34. Fluorescence of VLP in bothnative (i.e., Ab-free) and ZMapp™-treated AM appeared identical in bothsize and brightness, suggesting ZMapp™ did not induce agglutination(i.e., agglomeration of multiple Ebola VLP). Thus, the decrease inmeasured Ebola VLP mobility is most likely attributed to immobilizationof individual VLP due to polyvalent interactions between the array ofVLP-bound Ab and mucins.

To determine whether any particular mAb (c2G4, c116C6FR1, c4G7) withinthe ZMapp™ cocktail may confer superior “muco-trapping” potency, wemeasured the mobility of Ebola VLP in different aliquots of the same AMspecimens treated with the individual mAb. Interestingly, all three mAb,including one with Door neutralizing activity against Ebola, weresimilarly effective in reducing the mobility of Ebola VLP in AM, asshown in FIG. 16A. Relative to the control with no Ab and similar towith ZMapp™, the <D_(eff)> of Ebola VLP was reduced by ˜28-, 22-, and25-fold in AM treated with c2G4, c116C6FR1 and c4G7, respectively.Similarly, the fraction of mobile Ebola VLP was reduced to 27%, 33% and30%, respectively.

To better illustrate how the aforementioned changes in VLP mobilitymight alter the flux of virions reaching target cells, we performedfirst passage time analysis as described above. The predicted time for10% of Ebola VLP to diffuse across a 50 μm thick mucus layer increasedfrom ˜0.1 hours (i.e., approximately 5 minutes) for native AM to 2.2,2.1, 1.6, and 1.7 hours for ZMapp™-, c2G4-, c116C6FR1- and c4G7-treatedAM, respectively. Similarly, the estimated time for 50% of VLP to crossthe mucus layer increased from 0.5 hours for native AM to 23, 22, 20,and 22 hours, respectively. Since mucociliary clearance occurs on theorder of 15-30 minutes, these results suggest that, with the exceptionof native AM that is devoid of Ebola-binding IgG, the vast majority ofEbola virions would be quickly trapped and eliminated fromZMapp™-treated airways before they could penetrate AM.

The topical delivery of ZMapp™ may facilitate the rapid elimination ofEbola from the lung airways: Lastly, we sought to evaluate whetherZMapp™-induced trapping of viruses in mucus secretions ex vivo wouldtranslate to an improved barrier against Ebola penetration of AM andconsequently altered distribution in the lung airways in vivo. Using anaerosol delivery device (e.g., PennCentury microsprayer), weadministered ZMapp™ or PBS to the mouse lung, followed by addition offluorescent Ebola VLP 30 minutes later. In control mice treated, weobserved substantial red fluorescence associated with Ebola VLPthroughout the lung airways, including fluorescence indicative ofmucosal penetration and accumulation in the underlying airwayepithelium, as shown in FIGS. 17A and 17B. In contrast, far fewer EbolaVLP were present in the airways of mice treated with ZMapp™ (FIGS. 18C,18D), presumably since VLP were trapped in the luminal mucus and rapidlycleared. Indeed, fluorescence levels in lung tissues of ZMapp™-treatedmice were almost 7-fold lower than those in control mice, and only3.5-fold above background. These results are consistent with previousobservations that nanoparticles that bind to mucin mesh fibers areunable to penetrate the mucus layer and reach the underlying epithelium.

Ebola VLP readily penetrate fresh human AM secretions, but can beeffectively immobilized in AM by antigen-specific IgG (e.g., ZMapp™).Trapping in turn facilitated rapid elimination of Ebola VLP from themouse airways. Although Ebola is generally not considered an airbornepathogen, aerosol transmission of Ebola virus is biologically plausible.Ebola virus is present in saliva, feces, blood, and other body fluidsthat can be aerosolized through Ebola symptoms (e.g., coughing,vomiting, diarrhea) and through health care delivery (e.g., intubation,suctioning, delivery of nebulized medications). Large droplets from ahuman sneeze can travel up to 1-2 m, while smaller droplets can travelup to 6-8 m away within seconds to a few minutes. Studies have alsofound Ebola to survive in aerosol form for tens of minutes if not hours.Ebola can initiate infection in cells present in the respiratory tract,and fatal respiratory infection has been observed in guinea pigs andnon-human primates following intranasal and aerosol exposure.Furthermore, Ebola transmission has been shown between infected andhealthy macaques and between infected pigs and macaques without directphysical contact, likely via aerosol or droplet transmission, althoughpotential cross-contamination during animal husbandry practices couldnot be discounted entirely. A study frequently cited in argumentsagainst airborne transmission found no detectable Ebola transmissionwhen monkeys inoculated intramuscularly were housed in neighboringopen-barred cages separated by a Plexiglas® divider that preventeddirect contact; nevertheless, high viral titers were only detected inthe blood, suggesting virus titers in the lungs of these animals may nothave achieved the critical titers necessary for respiratory transmissionbefore the animals succumbed to the systemic effects of infection. Incontrast, recent studies in humans have reported substantial quantitiesof Ebola in respiratory secretions, and pathology studies have alsofound viral antigen in lung tissue. Finally, aerosol dissemination ofweaponized forms of these viruses, a version of which has reportedlyalready been developed for Marburg virus, presents a significant threatto both the military and potentially the general public. Altogether,these reports substantiate continued concern over aerosol transmissionof Ebola virus, and the need to explore strategies to prevent and treatEbola transmission at mucosal membranes, particularly if Ebola or otherfiloviruses become weaponized.

While the secretion of mucus can increase in response to infection,mucus is generally viewed as a passive rather than adaptive barrieragainst pathogens, and consequently overlooked in most studies ofmucosal infection. The notion that Ab can work in tandem with mucus toreinforce the diffusional barrier properties of mucus has remainedlargely unexplored, despite the fact that large quantities of Ab,including both IgG and IgA, are secreted into AM. Here, in goodagreement with our recent discovery that IgG can trap HSV-1 in humancervicovaginal mucus, we showed that Ebola-binding mAb, in the form ofZMapp™, were able to facilitate effective trapping of the majority ofEbola VLP in AM. Ab-mediated trapping of Ebola VLP markedly reduced thefraction of virions predicted to penetrate the mucus layer over thefirst few minutes of exposure. Since trapped viruses are quicklyeliminated by natural mucociliary clearance in the airways,ZMapp™-mediated trapping of Ebola would likely reduce the total flux ofviruses arriving at the airway epithelium and thus the likelihood and/orseverity of infection, rather than simply delaying the onset ofinfection. Consistent with this hypothesis, we observed topical delivery(e.g., via aerosol) of ZMapp™ into the mouse lung greatly reduced theamount of fluorescent Ebola VLP retained in the conducting airwayswithin 30 minutes. IgG-mediated trapping of viruses in mucus appears tobe a universal protective immune function across different mucosalsurfaces that enables protection directly at the portals of entry forviral transmission.

The concept of mucosal Ab prophylaxis and/or therapy based on Abdesigned to work together with mucus to trap pathogens represents aunique and complementary approach in the arsenal of protective methodsagainst infectious disease. First, the concept radically shifts thefirst line of defense against respiratory viruses to extracellular mucusgels instead of cellular targets, which is especially important againstviruses that are either exceptionally virulent (e.g., Ebola) and/orwithout a cure (e.g., HIV, Ebola). Second, Ab that trap viruses in mucusneed not bind to neutralizing epitopes; this greatly broadens thepotential antigen targets that can be exploited to achieve protection.Indeed, one of the mAb in the ZMapp™ cocktail is actually a poorneutralizer. Third, since the viral load during the transmission episodeat mucus membranes is likely low, the overall dose of mAb needed atmucosal surfaces, either before or immediately following a high-riskexposure event, may be substantially less than the mAb dose needed totreat a proliferating systemic infection. Thus, ZMapp™ deliveredtopically may be a particularly useful preventative measure or emergencyintervention for populations at the highest risks of acquiring Ebolainfections, such as healthcare workers, for reducing both the odds ofbecoming sick as well as the viral load entering the circulationfollowing an exposure event.

Example 6 Blocking Penetration of Respiratory Syncytial Viruses in HumanAirway Mucus

In the U.S., RSV is the leading cause of bronchiolitis, pneumonia andviral death, with >3.5 million RSV infections and >125,000hospitalizations in children under 2 each year. RSV infection in nursinghomes ranges between 5-10%, leading to 177,000 hospitalizations andsignificant rates of pneumonia (10-20%) and death (14,000 deathsannually) among the infected elderly. Palivizumab (also referred to asSYNAGIS) is a mAb developed for treatment of RSV. Despite its modestefficacy in reducing risk of RSV-associated hospitalization, SYNAGIS isonly given to a very small subset of the pediatric population. Per theCDC, there is currently no treatment for RSV.

SYNAGIS is typically applied by injection. Although it may decrease therisk of developing RSV, SYNAGIS is not effective in treating RSVinfection. Surprisingly, based on the inventions described herein, thisis likely because the amount of mAb extravasating into the airways isinadequate. In other words, there is likely just enough mAb reaching thelung lumen with IM injection to modestly reduce the rate of acquiringRSV infection, especially since the viral titers of typical RSV exposureare low. However, once infection is established, the amount of mAb isinadequate to contain the spread of higher titers of RSV. Given theunique pathophysiology of RSV that sheds infectious virions into thelumen, we believe concentrating mAb in the lung would be a moreeffective therapeutic approach than systemic or IM delivery. In cottonrat studies, pulmonary delivery of polyclonal IgG was 160-fold moreeffective in treating RSV than intramuscular delivery.

To visualize individual RSV virions in AM, we fluorescently tagged RSVby conjugating AF488 dye to the virus surface, which reduced the totalmAb that could bind RSV by only ˜10%-20%. When mixed into fresh,physiological human AM collected from intubated endotracheal tubes, asubstantial fraction of RSV (˜28%±7%) exhibited rapid diffusion in all12 independent AM specimens tested (FIGS. 19A-19B). The large fractionof trapped RSV (all RSV to the left of the dashed line) is likely due toendogenous RSV-binding Ab in adult AM; we expect the fraction of mobileRSV would be higher in AM from young children, who are less likely tohave substantial endogenous RSV-binding Ab. In aliquots of the same AMspecimens, we mixed into AM our “muco-trapping” palivizumab to a finalconcentration of 1 μg/mL, and found that virtually all RSV becameimmobilized (i.e., moved less than their diameter (d˜100 nm as indicatedby the dashed line) over at least the course of the 20 s movies). Weobserved similar trapping potency down to 50 ng/mL. Addition of anon-binding IgG control (a human IgG₁ against polyethylene glycol(PEG)), did not slow the diffusion of RSV. As described above, virionscan be effectively immobilized in human AM by virion-binding Ab.

The “muco-trapping” palivizumab described herein includes the heavy andlight chain variable regions of palivizumab and includes a glycosylationpattern on the Fc region that enhances mucous trapping. For example, themuco-trapping palivizumab may be a population of mAb in which at leastsome percentage (e.g., at least 50%, at least 60%, etc.) of the mAbhaving the heavy and/or light chain variable regions of palivizumab (ora derivative of palivizumab) contains a glycosolyation pattern of G0/G0F(i.e. GnGn) such that they facilitate trapping of viruses in mucus. Forexample, in some variations the population of muco-trapping antibody(e.g., muco-trapping palivizumab) has at least 60% of the recombinantantibodies in the population having a glycosylation pattern comprisingthe biantennary core glycan structureManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch.

Further, muco-trapping mAb against RSV rapidly eliminates RSV from mouseairways. To verify that trapping in mucus facilitates rapid eliminationfrom the airways, we performed an in vivo study where fluorescent RSVwas dosed to the airways of Balb/c mice (8-10 weeks), followed by eitherPBS or muco-trapping Ab against RSV, and finally euthanasia andcryo-sectioning of the trachea and airways 30 mins later. As shown bycryosectioned images, topical delivery of the RSV-trapping IgGeffectively reduces the amount of RSV remaining in the mouse airways aslittle as 30 mins post-delivery compared to PBS control, reaching levelscomparable to mouse treated with the same RSV-trapping IgG 15 mins priorto RSV inoculation (n=6; FIGS. 20A-20B). Similar results to PBS wereobserved with topical delivery of control IgG (n=2). These resultsunderscore the ability of muco-trapping IgG against RVS in enablingrapid elimination of RSV from infected airways.

Intranasally dosed “muco-trapping” mAb against RSV effectively reducesinfectious RSV titers in cotton rats. Based on our observations abovethat muco-trapping mAb against RSV was able to trap RSV in human AM andfacilitate rapid elimination of RSV in the mouse lung, we next proceededto evaluate whether topical delivery of the same mAb can be used totreat RSV infections. Cotton rats remains the gold standard as a firstin vivo model for RSV infection. In this study, we inoculated RSV StrainA2 (100 μL; 1×10⁶ PFU per rat) on Day 0. After a 48 hr period to allowthe infection to proliferate, we treated 3 groups of mice with either 2daily doses (i.e., once each on Day 2 and Day 3) of the muco-trappingmAb against RSV (possessing same Fab as SYNAGIS, modified to haveenhanced mucosal trapping as described herein; n=9), control IgG (e.g.,HGN-194, a mAb against HIV; n=8), or vehicle control (e.g., saline;n=8). All treatments were administered intranasally, and lungs wereharvested from all rats on Day 4, snap frozen, and finally homogenizedfor viremia analysis via immunoplaque assay. An anti-RSV antibody havingenhanced mucosal trapping (Muco-trapping mAb) mediated onaverage >100-fold reduction in infectious viral titers in the cotton ratlung, including 3 rats that showed no detectable viremia, underscoringthe potential effectiveness of this treatment. This is illustrated in,FIG. 21.

To demonstrate that we can deliver mAb to the lung airways vianebulization, we performed initial characterization of nebulizationusing PARI's off-the-shelf eRapid system. Using the Copley NextGeneration Impactor (NGI), shown in FIG. 22A. We tested the eRapiddevice operated at 15 L/min for 3 minutes using a human IVIG solution(Privigen®) at 25 mg/mL. Analysis of inertial impaction results, basedon distribution of antibody mass with respect to impaction stage cutoffdiameter, shows a Mass Median Aerodynamic Diameter (MMAD) of 4.5±0.21 μm(GSD=1.57±0.01), with a fine particle fraction of 66.5±6.2% (indicativeof the percentage of mass deposited on stage 3 of the NGI and belowwhich, at 15 L/min, has a cutoff diameter of 5.39 μm). These resultsunderscore our ability to generate aerosol droplets using a vibratingmesh nebulizer in a size range that support lung mucous deposition (1-5μm range). The MMAD of 4.5 μm matches well with the value presented bythe eFlow nebulizer manufacturer. To further validate that the IgG isstably nebulized, we performed gel electrophoresis on the nebulized IgG.We did not observe separation of the heavy and light chains oraggregation, and the concentration of IgG recovered was at 93%-108% ofthe input concentration. These results validate the ability to deliverby nebulizers.

Example 7 Nebulized mAb

Inertial Impaction and Aerodynamic Particle Size Distribution (APSD) wasdetermined by inertial impaction (NGI, MSP Corp., USA) operated at 15L/min for 3 minutes of actuation. No pre-separator was used and the NGIwas cooled for at least 90 minutes in a refrigerator to reduceevaporation of nebulized product during dosing. Privigen (CSL Behring)was added to the nebulizer (PART eFlow, as will described for FIG.23A-23C) at a concentration of 25 mg/mL and impaction trials wereperformed in triplicate. A custom mouthpiece adaptor was made frompolydimethylsiloxane (PDMS, Dow Corning). Deposited Privigen on eachimpactor stage was collected with 5 mL saline and quantified viaenzyme-linked immunosorbent assay (ELISA) using a Luminex MAGPIX(Austin, Tex.). This allowed for the determination of APSD andsubsequently mass median aerodynamic diameter (MMAD) by determining thecumulative mass percent undersize at each stage of the NGI plottedagainst the logarithm of the corresponding aerodynamic cutoff diameterand solving for the median particle size. Geometric size distribution(GSD) was calculated by the square root of the ratio of the particlesize at the 84th and 16th percentile of particle size distribution andfine particle fraction was defined as the proportion of IgG masscollected on stage 3 and below (particles <5.39 μm) relative to thetotal mass deposited. RSV-specific IgG were measured (see, e.g., DOI:10,1038/s41467-017-00739-6).

Although purified human or humanized monoclonal antibody (mAb) wasnebulized using a vibrating mesh nebulizer, any appropriate nebulizermay be used, including (but not limited to): jet nebulizer, vibratingnet nebulizer, etc. Further, although the methods and examples describedherein illustrate ‘wet’ nebulizer formulations, in some variationsdry/powder inhaler formulations may be used.

As shown in FIGS. 23A-23C, nebulized solutions (e.g., aerosol droplets)containing a recombinant antibody against an epitope in the A antigenicsite of the F protein of RSV that includes a population enriched foroligosaccharides having a glycosylation pattern that enhances thetrapping potency of the recombinant antibody in mucus may be formedhaving a droplet size that is appropriate for delivery into a patient'sairway (e.g., less than about 5 μm). In FIG. 23A, the mass medianaerodynamic diameter (MMAD) for this example is approximately 4.6 μm(GSD=2.2), and a mass percentage of aerosols below 5.4 μm of 53%, wasgenerated with IN-002 at a concentration of 15 mg/mL. In FIG. 23A, theemitted Dose is approximately 103%, and the fraction less than 5.39 μmis about 52.6%; the fraction less than 3.3 μm is about 30.1%. Theseaerosol characteristics are suitable for delivery into the lung airways.Importantly, IN-002 contained in the droplets appear to be stable, asindicated by total human IgG ELISA assays with coating antibody thatbinds IgG-Fab. The assay signal of nebulized IN-002 collected fromdifferent stages of the next generation impactor (NGI) were highlycomparable to unnebulized IN-002 control. Similarly, the antigenaffinity appears to be preserved, as shown by a whole-RSV ELISA assaybased on RSV-A2 coat and detection using an anti-human IgG secondaryhorseradish peroxidase conjugate, as shown in FIG. 23B. In FIG. 23C, thesignals of nebulized IN-002 collected from different stages of the NGIwere highly comparable to unnebulized IN-002 control.

Lower concentrations of mAb (e.g., mAb directed against respiratoryvirons, such as IN-001 and IN-002), were not as effectively delivered.For example, FIGS. 23D-23F show that nebulizing at lower concentrations,e.g., 5.4 mg/mL, led to substantially more loss of intact mAb asquantified by ELISA and also loss in binding affinity to viral antigencompared to a higher concentration of mAb, e.g., 15 mg/mL (as shown inFIGS. 23A-23C).

The Applicants have found that there appears to be an optimalconcentration range for treating or preventing Respiratory syncytialvirus (RSV) in a subject inhaling the nebulized solution. In particular,when the nebulized solution includes a recombinant antibody (e.g., suchas palivizumab, or a variant of palivizumab like motavizumab) having ahuman or humanized Fc region and an oligosaccharide having aglycosylation pattern that enhances the trapping potency of therecombinant antibody in mucus, wherein the nebulized solution comprisesparticles having a mass median aerodynamic diameter (MMAD). The optimalconcentration of antibody within particles in this MMAD size range isbetween about 10 mg/mL and about 100 mg/mL, specifically greater than 10mg/mL and less than 100 mg/mL (e.g., between 11 mg/mL and about 100mg/ml, between 12 mg/mL and 100 mg/mL, between 13 mg/mL and 100 mg/mL,between 14 mg/mL and 100 mg/mL, between 15 mg/mL and 100 mg/mL). Belowthis range, the treatment less effective or even ineffective. In somevariations this treatment is delivered and is optimized for deliveryonly after 2-3 days post-infection.

PARI LC Sprint™ nebulizers were used to administer RSV virus (e.g., 6 mLof RSV M37 strain at 1.1×10⁷ FFU/mL in media containing 20% sucrose) orcontrol media (media from HEp-2 cells lacking RSV) to individual lambs.Three 2-mL aliquots of virus-containing media or control media wereadministered to each animal over the course of 23 minutes resulting in atotal inhalation of about 3 mL by each lamb. The RSV-inoculated lambseach received a calculated effective dose of 0.1.29×10⁷ totalFFU/animal. Aerogen Solo nebulizers were used to deliver the nebulizedsolutions described above.

On either day 2 or day 3 post-infection, neonatal lambs were treatedwith either saline or two different muco-trapping mAb, IN-001 andIN-002. Nebulized IN-001 (beginning on day 3, and dosed also on day 4and 5) were delivered at a target inhaled dose of ˜0.9 mg/kg. NebulizedIN-002 (beginning on day 3, and dosed also on day 4 and 5) weredelivered at a target inhaled dose of ˜0.8 mg/kg. Nebulized IN-002 (lowdoses) were given beginning on day 2, and also on day 3, 4 and 5, at atarget inhaled dose of ˜0.3 mg/kg. Target inhaled doses were calculated(DOI: 10.1080/19420862.2018.1470727).

The thorax was opened, lungs removed and gross lesions were scored, asshown in FIGS. 24A-24E and 24F. The lungs were photographed in situ andex vivo. After removal, percentage parenchymal involvement was scoredfor each lung lobe before the BALF collection procedure. Left and rightlungs will be then separated and each lobe excised. Tissue samples werecollected from each lung lobe of all animals. In brief, one sample fromeach lobe not destined for BALF collection (i.e. 4 lobes—Right Cranial,Left Cranial, Left Middle and Left Caudal) were snap-frozen in liquidnitrogen for qRT-PCR. Two samples from each of these lobes were placedin tissue cassettes and put in 10% neutral-buffered formalin (NBF) forhistological and immunohistochemical analysis, and 1 representative lungsample (˜1 g) from each of these lobes was placed into 1 cryovial (1cryovial per lung sample—i.e. 4 cryovials per lamb) and immediatelysnap-frozen in liquid nitrogen, then transferred to −80° C. for storage.

As shown in FIGS. 24A-24E, the effectiveness of using nebulizedmuco-trapping mAb to treat RSV infection was assessed from theinoculation of infectious RSV (Memphis R37 strain) into 3-5 days oldlambs on Day 0, and initiated treatment on either Day 2 or Day 3post-infection. The lungs were harvested from the animals on Day 6, andthe gross lesions found on lung tissues, which reflect the extent of RSVinfections that occurred in the lungs of these animals, were scored. Asshown in FIGS. 24C-24E, pulmonary delivery of muco-trapping mAb (IN-001and IN-002) effectively reduced the gross lesions compared to salinecontrol (FIG. 24B). Indeed, many of the animals had no visibleRSV-induced lesions, and the lung pathology appeared indistinguishablecompared to the lungs in non-infected lambs.

Bronchoalveolar Lavage Fluid (BALF) samples from each animal werecollected immediately after euthanasia at day 6. Right caudal lobe BALFwas collected in 5 mL of cold double-modified Iscove's media (DMIM)containing 42.5% Iscove's modified Dulbecco's medium, 7.5% glycerol, 1%heat-inactivated FBS, 49% DMEM, and 5 μg/mL kanamycin sulfate. For BALF,immediately after lung dissection, the right caudal lung lobe from lambs1 to 20 were flushed through its major bronchus five times (with asingle 5 mL aliquot of DMIM) using a 10 mL serological pipet fitted toan electronic pipettor and about 2.6-3.0 mL were collected. 100 μL Rightcaudal lobe BALF was used for qRT-PCR assessment of RSV load by ISU and1.2 mL (from which 850 μL was used) for FFU analysis by ISU; asdescribed below. In addition, BALF was also collected from the rightmiddle lung lobe of each animal by flushing it five times (using a 1 mLpipet) through its major bronchus with five separate 1 mL aliquots ofsterile 0.9% saline, about 2-3.5 mL of which were recovered in mostcases.

Infectious focus-forming unit (FFU) assays on BALF and Lung Tissue wereperformed, the results of which are illustrated in FIG. 25. BALF samplesfrom each lamb were kept on ice in 1.5 mL vials and were used in astandard infectious focus assay as soon as possible after collection atnecropsy. Prior to use in the infectious focus assay, collected BALFsamples were first microfuged at 1780×g for 5 minutes to pellet anylarge debris, after which 800-850 μL of each clarified supernatant wereremoved by pipet (being certain to avoid debris on the bottom of eachtube; which can clog 0.45 μm filters) and applied and spun through an850 μL-capacity 0.45 μm Costar SPIN-X filter (Fisher Scientific, HanoverPark, Ill., USA) at 15,600×g for 5 minutes. At this point the BALFsamples were considered ready for infectious focus assay.

In addition, prior to use in the infectious focus assay, 0.5 g ofcollected lung samples (pooled from 0.1 g from each of 5 lobes for eachanimal; right cranial, accessory, left cranial, left middle, and leftcaudal) were immediately homogenized in 5 mL of DMIM on ice for 80seconds using an OMNI TH homogenizer. 1 mL of each homogenate wastransferred to a sterile 1.5 mL vial and microfuged at 1780×g for 5minutes to pellet large debris. 800-850 μL of each clarified supernatantwas removed by pipet and applied to a new SPIN-X column which was spunfor 5 minutes at 15,600×g. This was repeated two or three more times(using a new SPIN-X column each time) for each lung sample. At thispoint, the lung sample homogenates were considered ready for infectiousfocus assay.

Briefly, HEp-2 cells were grown to 70% confluence in 12-well cultureplates in DMEM media supplemented to 10% with heat-inactivated fetalbovine serum (FBS) and 50 μg/mL kanamycin sulfate. Collected BALFsamples were microfuged for 5 minutes at 3000×g to pellet large debris.850 μL of each supernatant was removed and spun through 850 μL-capacity0.45 μm Costar SPIN-X filters at 15,600×g for 5 minutes. Eachfilter-clarified BALF sample was analyzed at full-strength and at fouradditional serial-dilutions of 1:10, 1:100, 1:1,000 and 1:10,000. 200 μLof each sample dilution were added in duplicate to wells of a 12-wellplate (e.g.; each sample used an entire 12-well culture plate); twocontrol wells on each plate received virus-free cell culture medium.Plates will be incubated for 80 minutes in a CO2 incubator at 37° C. and5% CO2 with manual rocking every 20 minutes. 1 mL of culture medium wasadded to each well and cells were allowed to incubate for 48 hours afterwhich medium was removed and cells fixed with 60% acetone/40% methanolsolution for 1 minute. The fixing solution was removed and plates wereallowed to air-dry for 2 minutes after which each well was rehydratedwith 1 mL TBS-0.05% Tween 20, pH 7.4-7.6 (TBST) for 1 minute with mildrotation. To block non-specific binding, 1 mL of 3% BSA (FisherScientific, Hanover Park, Ill., USA) in TBST was added to all wells atroom temperature with gentle rocking for 30 minutes. Primary polyclonalgoat anti-RSV (all antigens) antibody (EMD Millipore, Billerica, Mass.)were diluted 1:800 in TBST containing 3% BSA; 325 μL of this was addedto each well and plates were allowed to incubate overnight at 4° C. withgentle rocking. The next day, plates were behed gently three times for 5minutes each with TBST, then 325 μL secondary antibody [Alexa Fluor® 488F (ab′) 2 fragment of rabbit anti-goat IgG (H+L), Molecular Probes/LifeTechnologies] diluted 1:800 in TBST containing 3% BSA was added to eachwell and allowed to incubate at room temperature for 30 minutes withgentle orbital rotation. Plates were rinsed two times for 5 minutes eachwith TBST and 1 mL of TBST was added back to each well prior tomicroscopic inspection. Plates were examined for the presence offluorescing infectious foci using the FITC/GFP filter on an invertedstage fluorescence microscope (Olympus CKX41, Center Valley, Pa., USA).Clusters of 5 or more fluorescing cells were counted as singleinfectious focal events. Titers will be calculated using the followingformula: an average of 20 counts in a 1:100-diluted (duplicate) sampleindicated that the original BALF sample had a “titer” of 10,000 since[20 counts×dilution factor of 100×1,000 μL/mL]/200 μL assessed=10,000infectious focus-forming units/mL (FFU/mL). For lung homogenatecalculations: an average of 20 counts in a 1:100-diluted sampleindicates that the original lung sample had a titer of 110,000 FFU/glung tissue since 11×[20 counts×dilution of 100×1000 μL/mL]/200 μLassessed=110,000 infectious foci “FFU”/g. These results will bemultiplied by fraction of sample loss

As shown in FIG. 25, a quantitatively assess the infectious viral loadpresent in the lungs of lambs at the time of sacrifice was examined by astandard focus-forming unit assay either on lung homogenates. FIG. 26shows bronchial alveolar lavage fluid (BALF). In lung homogenates,either markedly reduced FFU (IN-001) or no detectable FFU was observed(when treated with IN-002, either at full dose starting on Day 3 orreduced dose starting on Day 2). Similar results were found in BALFobtained from the same lambs, with treatment using IN-002 reducing theinfectious viral load by nearly 4 orders of magnitude. These resultsunderscore the effectiveness in delivering muco-trapping mAb fortreatment of RSV.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A nebulized solution for treating or preventing Respiratory syncytialvirus (RSV) in a subject inhaling the nebulized solution, the nebulizedsolution comprising a recombinant antibody having a human or humanizedFc region and an oligosaccharide having a glycosylation pattern thatenhances the trapping potency of the recombinant antibody in mucus, sothat the recombinant antibody binds to the RSV to form an antibody/RSVcomplex that is trapped in the subject's mucus thereby treating orpreventing the infection, wherein the nebulized solution comprisesparticles having a mass median aerodynamic diameter (MMAD) of between2-6 μm diameter and a concentration of antibody within the particles ofbetween 10 mg/mL and 100 mg/mL.
 2. The nebulized solution of claim 1,wherein the pH of the nebulized solution between about 4.5 to
 7. 3. Thenebulized solution of claim 1, wherein the pH of the nebulized solutionapproximately neutral.
 4. The nebulized solution of claim 1, wherein thenebulized solution is hypertonic relative to the lungs.
 5. The nebulizedsolution of claim 1, wherein the recombinant antibody comprises anN-linked glycosylation site on the Fc region of the antibodies to whichthe oligosaccharide is attached.
 6. The nebulized solution of claim 1,wherein the glycosylation pattern comprises a biantennary core glycanstructure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch.
 7. The nebulized solution of claim1, wherein the recombinant antibody comprises a human or humanized IgGor IgM monoclonal antibody, or a fragment or derivative thereof.
 8. Thenebulized solution of claim 1, wherein the recombinant antibodycomprises palivizumab, or a variant of palivizumab.
 9. The nebulizedsolution of claim 1, wherein the recombinant antibody comprisesmotavizumab.
 10. The nebulized solution of claim 1, further comprising asurfactant.
 11. The nebulized solution of claim 1, wherein theconcentration of antibody within the particles of between 12 mg/mL and85 mg/mL.
 12. A nebulized solution for treating or preventingRespiratory syncytial virus (RSV) in a subject inhaling the nebulizedsolution, the nebulized solution comprising a recombinant antibodyagainst an epitope of the F protein of RSV, the recombinant antibodyhaving a human or humanized Fc region and an oligosaccharide having aglycosylation pattern that enhances the trapping potency of therecombinant antibody in mucus, so that the recombinant antibody binds tothe RSV to form an antibody/RSV complex that is trapped in the subject'smucus thereby treating or preventing the infection, wherein thenebulized solution comprises particles having a mass median aerodynamicdiameter (MMAD) of between 2-6 μm diameter, a pH between 4.5 and 7, anda concentration of antibody within the particles of between 12 mg/mL and100 mg/mL.
 13. The nebulized solution of claim 12, wherein the nebulizedsolution is hypertonic relative to the lungs.
 14. The nebulized solutionof claim 12, wherein the recombinant antibody comprises an N-linkedglycosylation site on the Fc region of the antibodies to which theoligosaccharide is attached.
 15. The nebulized solution of claim 12,wherein the glycosylation pattern comprises a biantennary core glycanstructure of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch.
 16. The nebulized solution of claim12, wherein the recombinant antibody comprises a human or humanized IgGor IgM monoclonal antibody, or a fragment or derivative thereof.
 17. Thenebulized solution of claim 12, wherein the recombinant antibodycomprises palivizumab, or a variant of palivizumab.
 18. The nebulizedsolution of claim 12, wherein the recombinant antibody comprisesmotavizumab.
 19. The nebulized solution of claim 12, further comprisinga surfactant.
 20. The nebulized solution of claim 12, wherein theconcentration of antibody within the particles of between 12 mg/mL and85 mg/mL.
 21. A nebulized solution for treating or preventing arespiratory virus in a subject inhaling the nebulized solution, thenebulized solution comprising a recombinant antibody having a human orhumanized Fc region and an oligosaccharide having a glycosylationpattern that enhances the trapping potency of the recombinant antibodyin mucus, so that the recombinant antibody binds to the respiratory toform an antibody/respiratory virus complex that is trapped in thesubject's mucus thereby treating or preventing the infection, whereinthe nebulized solution comprises particles having a mass medianaerodynamic diameter (MMAD) of between about 2 and 6 μm diameter and aconcentration of antibody within the particles of between 10 mg/mL and100 mg/mL.
 22. The nebulized solution of claim 21, wherein therespiratory virus is one of: Respiratory syncytial virus (RSV),metapneumovirus, influenza virus, adenovirus, and parainfluenza.
 23. Thenebulized solution of claim 21, wherein the pH of the nebulized solutionbetween 4.5 and
 7. 24. The nebulized solution of claim 21, wherein thenebulized solution is hypertonic relative to the lungs.
 25. Thenebulized solution of claim 21, wherein the recombinant antibodycomprises an N-linked glycosylation site on the Fc region of theantibodies to which the oligosaccharide is attached.
 26. The nebulizedsolution of claim 21, wherein the glycosylation pattern comprises abiantennary core glycan structure ofManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1 with terminalN-acetylglucosamine on each branch.
 27. The nebulized solution of claim21, wherein the recombinant antibody comprises a human or humanized IgGor IgM monoclonal antibody, or a fragment or derivative thereof.
 28. Thenebulized solution of claim 21, wherein the concentration of antibodywithin the particles of between 12 mg/mL and 85 mg/mL.